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Cite this: RSC Adv., 2020, 10, 15154

Strategic design to create HER2-targeting proteins
with target-binding peptides immobilized ona ﬁbronectin type III domain scaﬀold†
Wanaporn Yimchuen,a Tetsuya Kadonosono, *a Yumi Ota,a Shinichi Sato,
Maika Kitazawa,a Tadashi Shiozawa,a Takahiro Kuchimaru, c Masumi Taki,
Yuji Ito,e Hiroyuki Nakamura b and Shinae Kizaka-Kondoh abdTumor-binding peptides such as human epidermal growth factor receptor 2 (HER2)-binding peptides are
attractive therapeutic and diagnostic options for cancer. However, the HER2-binding peptides (HBPs)
developed thus far are susceptible to proteolysis and lose their aﬃnity to HER2 in vivo. In this report,
a method to create a HER2-binding ﬂuctuation-regulated aﬃnity protein (HBP-FLAP) consisting ofa ﬁbronectin type III domain (FN3) scaﬀold with a structurally immobilized HBP is presented. HBPs wereselected by phage-library screening and grafted onto FN3 to create FN3-HBPs, and the HBP-FLAP withthe highest aﬃnity (HBP sequence: YCAHNM) was identiﬁed after aﬃnity maturation of the grafted HBP.
HBP-FLAP containing the YCAHNM peptide showed increased proteolysis-resistance, binding to HER2
Received 15th January 2020
Accepted 5th April 2020

with a dissociation constant (KD) of 58 nM in ELISA and 287 nM in biolayer interferometry and speciﬁcally

DOI: 10.1039/d0ra00427h

detects HER2-expressing cancer cells. In addition, HBP-FLAP clearly delineated HER2-expressing tumors
with a half-life of 6 h after intravenous injection into tumor-bearing mice. FN3-based FLAP is an

rsc.li/rsc-advances

excellent platform for developing target-binding small proteins for clinical applications.

1. Introduction
The human epidermal growth factor receptor 2 (HER2) isa member of the human epidermal growth factor receptor(HER/EGFR/ErbB) family of receptor tyrosine kinases that plays
vital role in human cancer.1 HER2 is a therapeutic target
because of its role in the progression of many cancers, including
breast cancer,2 gastric cancer3 and ovarian cancer.4 Accordingly,
HER2-targeting monoclonal antibodies (mAbs) have alreadybeen approved by the Food and Drug Administration for treatment of HER2-positive cancers: trastuzumab for the treatmentof breast cancer and gastric cancer;5 and pertuzumab fortreatment of HER2-positive breast cancer.6 These mAbs showhigh stability in the body and eﬀective treatment; however, theiraSchool of Life Science and Technology, Tokyo Institute of Technology, Yokohama
226-8501, Japan. E-mail: tetsuyak@bio.titech.ac.jp; Fax: +81-45-924-5848; Tel: +8145-924-5848

bInstitute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8501,
Kanagawa, Japanc

Center for Molecular Medicine, Jichi Medical University, Shimotsuke 329-0498, Japand

Graduate School of Informatics and Engineering, The University of ElectroCommunications, Tokyo 182-8585, Japane

Graduate School of Science and Engineering, Kagoshima University, Kagoshima 8900065, Japan† Electronic supplementary
10.1039/d0ra00427h

information

15154 | RSC Adv., 2020, 10, 15154–15162(ESI)

available.

See

DOI:

relatively large molecular weight (150 kDa) limits tissue penetration and production costs are high.7
Tumor-binding peptides are a promising alternative to mAbs
as they demonstrate greater tumor penetration because of theirsmall molecular size.8 Various kinds of HER2-binding peptides(HBPs) have been identied, including a linear peptide
KCCYSL,9 bicyclic peptides ACYLQDPNCDWWGPYCGGSG10
and a trastuzumab-derived cyclic peptide FCGDGFYACYMDV.11
Trastuzumab- and pertuzumab-derived peptides, QDVNTAVAW
and EWVADVNPNSGGFIYNQYFK, respectively, have also been
designed and used in tumor imaging.12,13 However, their clinicalapplication is limited to use as diagnosis probes because short
peptides are very sensitive to protease activity in circulation andtissues, resulting in reduced binding to targets. Therefore,
proteins with proteolysis-resistant target-binding peptides may
increase the applicability of tumor-binding peptides for clinicaluse.
Several strategies have been developed to increase
proteolysis-resistance of peptides in vivo, including conjugationof peptides to macromolecules, including polyethylene glycol(PEG), the Fc domain, or serum albumin.14 Cyclization of
peptides or graing of peptide into cyclic peptide are also
simple strategies to increase resistance to proteases.15,16
Recently, we found that the aﬃnity and proteolysis-resistance of
peptides was greatly improved aer their structure was immobilized by graing into a particular site of a protein scaﬀold.17
Based on this nding, we reported a computational design

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strategy for developing antibody mimetics named FLAP, whichis uctuation-regulated aﬃnity protein (FLAP) having structurally immobilized mAb-derived peptide.18
In this study, we focused on the development of a HER2targeting small protein with a structurally immobilized HBP,
HBP-FLAP. The optimal HBP in an HBP-FLAP showed improved
proteolysis resistance and exhibited specic HER2-binding invitro and in vivo. These results provide new routes for developing therapeutically applicable target binding proteins.

2.

Experimental section

2.1

Peptide screening

The T7 phage libraries displaying the random cyclic peptides
X3CX8CX3 or X3CX9CX3, where X represents the randomized
amino acids, were constructed using the T7 Select10-3b vector(Merck Millipore, Burlington, MA, USA), as described previously.19 HER2-Fc (R&D systems, Minneapolis, MN, USA) was
biotinylated using a labeling kit (Dojindo, Kumamoto, Japan)
and immobilized on streptavidin-conjugated magnetic beads(Tamagawa Seiki, Nagano, Japan). The beads were blocked with
0.5% BSA in PBS, incubated with the phage libraries of
X3CX8CX3 (1  1010 pfu) or X3CX9CX3 (1  1010 pfu) for 1 h,washed 30 times with PBS with 1% Tween 20 (1% PBST) andthen added to 7 mL of E. coli BLT5403 cells (Merck Millipore) inlog phase growth. Aer incubation at 37  C and bacteriolysis,
the phages were recovered from the culture supernatant by
centrifugation (9100 g for 10 min at 4  C) and used for the next
round of biopanning. Amino acid sequences of displayed cyclic
peptides on selected T7 phage clones were analyzed by DNA
sequencing.
2.2

ELISA

For phage-ELISA, the wells of 96-well black plates (Thermo
Fisher Scientic, Waltham, MA, USA) were coated with 1000 ng
of HER2-Fc, streptavidin (Wako, Osaka, Japan), or IgG1-Fc (R&D
systems) and blocked with 2% Perfect-Block (MoBiTec,
Göttingen, Germany) in PBS for 1 h. Phages (1  1010 pfu) wereadded to each well and incubated for 1 h. Aer washing theplate, the bound phages were detected with the anti-T7 ber tailantibody (Merck Millipore) as a primary antibody and the antimouse IgG HRP-linked antibody (Cell Signaling Technology,
Danvers, MA, USA).
For indirect ELISA, the wells were rst coated with 50 ng of
HER2-Fc and then blocked with 2% Perfect-Block in PBS for 2 h.
Aer incubation with sample proteins for 1 h, the HRPconjugated anti-His-tag antibody (Abcam, Cambridge, MA,
USA) was incubated for 1 h with the samples and then treatedwith the QuantaRed Enhanced Chemiuorescent HRP
Substrate kit (Thermo Fisher Scientic). The resulting uorescence was measured using an Innite F500 (Tecan, Mannedorf,
Switzerland) with specic lters (Ex/Em ¼ 535 nm/590 nm).
For specicity evaluation, the wells were incubated overnightwith HER2-Fc (50 ng/50 mL in PBS), EGFR-Fc (50 ng/50 mL in
PBS; R&D systems), or 50 mL PBS, and applied to the indirect
ELISA described above.

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RSC Advances
2.3

Molecular dynamics (MD) simulations

Initial coordinates of the bronectin type III domain (FN3)protein were taken from the Protein Data Bank (PDB ID: 1TTG).
The structures of linear peptides and FN3 mutants weregenerated using Discovery Studio 3.1 (Accelrys, San Diego, CA,
USA). The systems were optimized via energy minimization andequilibrated with backbone restraints. Production runs were
performed for at least 10 ns for trajectory analysis. All MD
simulations were performed using the Amber 14 and 16
program packages20 on TSUBAME (Global Scientic Information Center, Tokyo Institute of Technology, Japan). The Amber
ﬀ14SB force elds and the GB/SA implicit solvent model wereused. The time-step for MD simulations was set to 2 fs with the
SHAKE algorithm. A nonbonded cutoﬀ of 999.9 Å was used. The
temperature was kept constant at 300 K using the Berendsen
rescaling method. The average root-mean-square uctuation(RMSF) of all hydrogen-free atoms during the nal 5 ns of eachproduction run were calculated to investigate the backbone
uctuations in each system using the cpptraj module.
2.4

Plasmid construction and protein purication

The recombinant DNA experiment was carried out according to
the necessary regulations based on the Tokyo Institute of Technology recombinant DNA experimental safety management
regulations dened by the Tokyo Institute of Technology
recombinant DNA experimental safety management committee.
The cDNA encoding the fusion protein, consisting of a His-tag,
GGGS linker and FN3 (H-FN3), was inserted into the multicloning site of the pGEX-6P-3 plasmid (GE Healthcare, Little
Chalfont, UK). The cDNA encoding H-FN3 mutants, C-terminally
CTag (GMAQIEVNCSNE)21-fused His-tagged FLAP (H-FLAP-C), or
C-terminally CTag-fused H-FN3 (H-FN3-C) were constructed bysite-directed mutagenesis using the H-FN3 cDNA as the template.
The plasmid vectors expressing H-FN3, H-FN3 mutants, H-FN3-C
and H-FLAP-C were transformed into E. coli BL21 (DE3) pLysS
cells (Promega, Fitchburg, WI, USA). These proteins wereexpressed in the bacteria as GST fused proteins. GST-fused HFN3, H-FN3 mutants, H-FN3-C and H-FLAP-C were puriedfrom the supernatants of bacterial extracts using glutathione
agarose (Sigma-Aldrich, St Louis, MO, USA). The monomericpuried proteins were passed through Superdex 200 Increase 10/
300 GL columns (GE Healthcare), according to the manufacturer's instructions, to increase protein sample purity.
2.5

Cell immunostaining

The human cervical cancer cell line HeLa, human neuroblastoma
cell line SH-SY5Y, and human breast cancer cells expressing reyluciferase SK-BR-3 were obtained from the RIKEN Bio-Resource
Center (Tsukuba, Japan). The human gastric cancer cell line
N87 was obtained from the JCRB Cell Bank (Osaka, Japan). Cells
were maintained with 5% fetal bovine serum-Dulbecco's Modied
Eagle's Medium (Nacalai Tesque, Kyoto, Japan) supplemented
with penicillin (100 U mL1) and streptomycin (100 mg mL1)(Nacalai Tesque) in a 5% CO2 incubator at 37  C and regularly
checked for mycoplasma contamination by a mycoplasma test kit

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RSC Advances(Lonza, Basel, Switzerland). The cells (3.0  104 cells per well) wereseeded on a slide chamber plate, cultured for 16 h and xed bytreatment with a 4% paraformaldehyde solution for 10 min at
25  C. The cells were then blocked with 1% BSA-PBS for 1.5 h atroom temperature. Aer the addition of 1 mM of puried proteinsto the chamber, the cells were incubated for 16 h at 4  C. The Alexa
Fluor 488-conjugated mouse anti-His tag secondary antibody(Medical & Biological Laboratories, Aichi, Japan) was then used touorescently label the His-tagged proteins. The slide was mounted with mounting medium (DBS, Pleasanton, CA, USA) with 1/
1000 of Hoechst 33342 (Enzo Life Sciences, Farmingdale, NY,
USA). All photos were taken using a BZ-X700 microscope (Keyence,
Osaka, Japan) with the lters of BZ-X lter GFP, OP-87763 (Ex: 470/
40 nm, Em: 525/50 nm) and BZ-X lter DAPI, OP-87762 (Ex: 360/
40 nm, Em: 460/50 nm).
2.6

Flow cytometry

Single cell suspensions of HeLa, SH-SY5Y, SK-BR-3 and N87
cells (1.0  105 cells/100 mL) were incubated in PBS containing
1000 times-diluted Alexa Fluor 488 anti-human HER2 antibody(clone 24D2; BioLegend, San Diego, CA, USA) on ice in the darkfor 30 min. Cells without immunostaining were used asa negative control. Flow cytometry was performed by a owcytometer ec800 (Sony Biotechnology, San Jose, CA, USA).
2.7

Paper
Tween-20 in PBS) was used to load the ligand onto the surface of
streptavidin biosensors (ForteBio) for 300 s. Aer washing (30 s)
and equilibrating (60 s) the biosensor, the association of theligand on the biosensor to the analyte in solution (100–800 nM
His-tagged HBP-FLAP in the kinetic buﬀer) was measured for
300 s. The dissociation of the interaction was subsequently
measured for 300 s. Systematic baseline dri correction was
done by subtracting the shi recorded for sensors loaded withligand but incubated without analyte. Data analysis and curvetting were done using Octet soware version 11.0 (ForteBio).
Experimental data were tted with the binding equations
available for a 1 : 1 interaction with local tting and the mean standard error of the mean (SEM) values of kon and koﬀ were
calculated from the data of four diﬀerent concentrations ofanalytes. The KD was calculated as the ratio of koﬀ/kon.

2.10

Mice and in vivo uorescent imaging

All animal experiments were performed with the approval of theAnimal Ethics Committees of Tokyo Institute of Technology (no.
2017004) and in accordance with the Ethical Guidelines for
Animal Experimentation of Tokyo Institute of Technology. MaleBALB/c nude mice were purchased from Oriental Yeast Co., Ltd.
(Tokyo, Japan). All mice used were 6–9 weeks of age. For theProteinase K treatment

Fiy mM of FN3-p1.M.2 or the C-terminally elongated p1.M.2
peptide (YCAHNM-GSGSGK) was digested by 50 mU mL1
proteinase K for 2 h in 10 mM Tris–HCl (pH 8.0) containing
10 mM EDTA. Sample aliquots were collected at various reaction
times and analyzed by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) or reversed-phase highperformance liquid chromatography (HPLC; InertSustainSwi
C18 column; GL Sciences, Tokyo, Japan). The ratios of intact
FN3-p1.M.2 and the C-terminally elongated p1.M.2 peptide
were calculated based on the band intensity of the digested
products on the SDS-PAGE gel and the HPLC peaks in thechromatogram, respectively.
2.8

Preparation of HBP-FLAP and FN3 probes

HBP-FLAP-IR800 and FN3-IR800 monomer probes wereprepared by covalent conjugation of a uorescent dye IRDye
800CW-maleimide (LI-COR Biosciences, Lincoln, NE, USA) toa cysteine residue at the C-terminus of the CTag of H-FLAP-C.
Twenty-ve nmol of unlabeled H-FLAP-C or H-FN3-C was reacted with 50 nmol of IRDye 800CW-maleimide in PBS for 2 h atroom temperature. PD-10 desalting columns (GE healthcare)
were used to purify the reactants.
2.9

Biolayer interferometry

The kinetics of HBP-FLAP binding to HER2 was studied usinga ForteBio Octet Red (ForteBio, CA, USA) instrument. The assays
were performed at 30  C in 96-well black plates. HER2-Fc wasbiotinylated using a biotin labelling kit (Dojindo), and 100 nM
biotinylated HER2-Fc in the kinetic buﬀer (0.1% BSA, 0.002%

15156 | RSC Adv., 2020, 10, 15154–15162

Fig. 1 Overview of HBP-FLAP development by grafting HBP intoa small protein scaﬀold for peptide stabilization. (A) The HBPs wereisolated from a phage-displayed cyclic peptide library. (B) Hexapeptides trimmed from HBPs were grafted into the GA site of the FN3
scaﬀold to generate FN3-HBPs. (C) Binding aﬃnity of FN3-HBP was
improved by aﬃnity maturation using alanine scanning and the FN3HBP with the highest aﬃnity was screened using a phage-displayed
FN3-HBP mutant library to create the optimal HBP-FLAP. (D) The
aﬃnity-matured HBP-FLAP was conjugated with near infrared ﬂuorescence dye IRDye 800CW (IR800) to give an in vivo imaging probe,
HBP-FLAP-IR800.

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RSC AdvancesFig. 2 Preparation of HBP-FN3s. (A) The frequency of detection of HBPs during the phage library screening. Phage clones P1 and P2 wereselected from an eight-amino acid cyclic peptide (X3CX8CX3) library. Phage clones P3–P5 were selected from a nine-amino acid peptide(X3CX9CX3) library. (B) The binding aﬃnity of the indicated phage clones to HER2-Fc (grey), streptavidin (SA, black) and IgG-Fc (white) was
measured by ELISA and the relative aﬃnities to the control cyclic peptide library (Lib) are shown. n ¼ 3, mean  SEM, *p < 0.05 compared with Lib
based on a Student's t-test. (C) Amino acid sequence of the FN3 domain. The HBP was grafted into FN3 by substitution at residues 51–56, which
are highlighted in pink. (D) RMSF calculation of HBPs in linear form (grey) and grafted into the FN3 scaﬀold (black).

subcutaneous xenogra model, N87 cells suspended in PBS (1.0
 106 cells/20 mL) were mixed with an equal volume of Geltrex(Life Technologies) and injected into the forefoot of mice. Micewith subcutaneous tumors of 10–15 mm in diameter were used
for experiments. To acquire uorescence images, 300 pmol in
100 mL PBS of HBP-FLAP-IR800 or FN3-IR800 was intravenously
injected into mice with subcutaneous tumors in the forefoot.
The following conditions were used for image acquisition:
excitation lter ¼ 710  15 nm; emission lter ¼ 800  10 nm;
and exposure time ¼ 1 s. The minimum and maximum photons
per s per cm2 per sr of each image are indicated in each gure bya yellow-red bar scale.
2.11

Statistical analysis

Data in this study are expressed as means  standard error of themean (SEM). Diﬀerences are analyzed by a two-sided Student's ttest; p values of <0.05 were considered as statistically signicant.

3.

Results

3.1

Strategy for creating HBP-FLAP

Our previous research demonstrated that short peptides graedin a particular region of protein scaﬀold were structurallyThis journal is © The Royal Society of Chemistry 2020

Table 1

Amino acid sequences of the HER2-binding peptides

PeptidesAmino acid sequences

p1
p2
p3
p4
p5
p4.1
p4.2
p4.3
p4.4
p5.1
p5.2
p5.3
p5.4
p1.1A
p1.2A
p1.3A
p1.4A
p1.5A
p1.6A
p1.M.1
p1.M.2
p1.M.3YCGCEM
PFICCRRPRGRRCEHG
SLFCWVRVM
AMVCTRARKPKSGCRRVG
VELCATAKHEVKSCLSFGTRARKPRARKPK
ARKPKSRKPKSGATAKHE
TAKHEVAKHEVKKHEVKSACGCEMYAGCEM
YCACEMYCGAEM
YCGCAMYCGCEAYCICCMYCAHNMYCPVCM

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immobilized and showed increased resistance to proteases.17
This nding prompted us to develop clinically applicable HER2targeting small proteins with a structurally immobilized HBP.
Fig. 1 shows the process for producing such protein HBP-FLAP.
FN3, one of three types of internal repeats found in the plasma
protein bronectin, was selected as the scaﬀold because it is an
endogenous protein domain with high stability and high solubility.22,23 In addition, previous calculations revealed that residues 51–56 of FN3 are a gra acceptor (GA) site in which thestructure of graed hexapeptides can be immobilized.18 FN3
has been used for the development of aﬃnity proteins targeting
various antigens by protein engineering,23,24 some of which haveentered clinical trials,25 suggesting that FN3 is a promising
scaﬀold for creating HBP-FLAP.
The HBPs were rst isolated from a phage-displayed peptide
library (Fig. 1A) and then some six consecutive amino acids

Paper(hexapeptides) in the isolated HBPs were selected and graedinto the GA site of FN3 to create FN3-HBPs (Fig. 1B). The
binding aﬃnity of the FN3-HBPs was improved by aﬃnity
maturation (Fig. 1C). The aﬃnity-matured HBP-FLAP thatshowed the highest binding aﬃnity among the tested FN3-HBPs
was labeled with the near-infrared uorescence dye IRDye
800CW and evaluated for target-binding ability in vitro and invivo (Fig. 1D).

3.2

Design of a HER2-binding small proteins

Five T7 phage clones, P1–P5, displaying HBPs were enriched byeight rounds of biopanning against HER2-Fc from T7 phagelibraries displaying random cyclic peptides X3CX8CX3 or
X3CX9CX3, where X represents the randomized amino acids(Fig. 2A). Further evaluation of the phage clones by phage-ELISA

Aﬃnity maturation of HER2-binding small proteins. (A) ELISA was used to measure the aﬃnity of FN3-HBP mutants toward HER2-Fc. The
mean  SEM of three independent experiments is shown; *p < 0.05. (B) ELISA measurements showing the aﬃnity of alanine substitution mutantsof FN3-p1 toward HER2-Fc. The mean  SEM of three independent experiments is shown; *p < 0.05. (C) The pie chart shows the frequency ofdetection of the HBP sequence (YCXXXM) in screening after aﬃnity maturation. (D) Fluctuation of the residues of the indicated HBP sequences(YCXXXM) in FN3-p1. The RMSF values of each residue are shown. (E) Predicted structures of FN3, FN3-p1, and FN3-p1.M.1–M.3 showing the GAsite (pink) and the grafted HBPs (blue).

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RSC Advances

revealed their target specicity: P1, P4 and P5 bind strongly andspecically, whereas P2 binds weakly to HER2-Fc. P3 binds to
IgG-Fc non-specically (Fig. 2B). Therefore, HBPs expressed in
P1, P4 and P5 (hereaer named p1, p4 and p5) were used for thedevelopment of FN3-HBPs. Continuous hexapeptides p4.1–p4.4
and p5.1–p5.4 were prepared from p4 and p5, respectively(Table 1), and graed into the GA site of FN3 (Fig. 2C). Note that
p1 was originally displayed as a hexapeptide on T7 phage
because the seventh codon is a stop codon. Computational
evaluation by MD simulations conrmed a decrease in the
average RMSF value. This indicates that all linear hexapeptides
were immobilized in the GA site of FN3 (Fig. 2D). As a result, we
obtained nine FN3-HBPs: FN3-p1, FN3-p4.1, FN3-p4.2, FN3p4.3, FN3-p4.4, FN3-p5.1, FN3-p5.2, FN3-p5.3 and FN3-p5.4.

3.3

Aﬃnity maturation of HER2-binding small proteins

Recombinant FN3-HBPs were puried by a bacterial protein
expression system and used to determine the FN3-HBP withhighest aﬃnity to HER2. Two out of the nine FN3-HBPs, FN3p4.1 and FN3-p5.2, were not expressed but the other seven
FN3-HBPs were expressed successfully as soluble proteins andeasily puried. Evaluation of the binding aﬃnity to HER2
revealed that three out of the seven, FN3-p1, FN3-p4.3 and FN3p4.4 bound to HER2 (Fig. 3A) with dissociation constants (KD)
280–500 nM (Table 2, ESI Fig. S1A†), indicating moderatebinding aﬃnity. Sequence optimization of p1 with the lowest KD
value (KD ¼ 280 nM) was performed to increase the aﬃnity of
FN3-p1. By single amino acid substitutions in p1 (YCGCEM) toalanine (A), six FN3-p1 mutants, FN3-p1.1A, FN3-p1.2A, FN3p1.3A, FN3-p1.4A, FN3-p1.5A and FN3-p1.6A were generated(Table 1). Reduced binding aﬃnity to HER2 was observed with
FN3-p1.1A, FN3-p1.2A and FN3-p1.6A (Fig. 3B), indicating thatthe rst (Y51), second (C52) and sixth (M56) residues were
important for HER2 binding. Optimization of the third, fourthand h residues of p1 was performed using a T7 phagedisplayed FN3-p1 mutant library with three randomized
sequences 51-YCXXXM-56. Aer eight rounds of biopanning,
three peptides were selected from the most frequently enriched
FN3-p1 mutants: FN3-p1.M.1 (YCICCM), FN3-p1.M.2(YCAHNM) and FN3-p1.M.3 (YCPVCM; Fig. 3C and Table 1).
Amino acid substitutions made in the third, fourth and hresidue positions showed minimal eﬀect on structural uctuations of Y51, C52 and M56 (Fig. 3D and E). Among the threeaﬃnity-matured FN3-p1 mutants, FN3-p1.M.2 showed theTable 2

Dissociation constants (KD) of the FLAP candidates

FN3-HBPs

KD (nM)

FN3-p1FN3-p4.3
FN3-p4.4
FN3-p1.M.1
FN3-p1.M.2
FN3-p1.M.3

280  23450  74500  150
68  1458  5.0
110  28This journal is © The Royal Society of Chemistry 2020

Fig. 4 Proteolysis-resistance of the p1.M.2 peptide and binding

speciﬁcity of HBP-FLAP to HER2-expressing cells. (A) Cleavage sites ofthe p1.M.2 peptide by proteinase K. Peptide bonds adjacent to thecarboxyl group of the 1st (Y) and 3rd (A) residues are cleaved (redarrows). (B) Proteinase K resistance of p1.M.2 peptides. Proteinase Ktreated FN3-p1.M.2 and the C-terminally elongated p1.M.2 peptide (cp1.M.2) were analyzed at 10, 30, 60, 90 and 120 min. Percentage ofintact FN3-p1.M.2 and the C-terminally elongated p1.M.2 peptide wascalculated by SDS-PAGE and HPLC analysis, respectively. n ¼ 3, mean
 SEM, *p < 0.05 compared with incubation time at 0 min based ona Student's t-test. (C) Flow cytometry analysis of HER2 expression on
the cell surface of HeLa (black line), SH-SY5Y (gray line), SK-BR-3 (blueline), and N87 (magenta line) cells. Histograms are representative of
duplicate samples. (D) Evaluation of HER2-binding aﬃnity of HBPFLAP to HeLa, SH-SY5Y, SK-BR-3 and N87 cells. HBP-FLAP and control
FN3 were labeled with AF488 (green) and reacted with ﬁxed cells. The
nuclei were stained with Hoechst (blue). Scale bar ¼ 50 mm. (E) Speciﬁc
binding of HBP-FLAP to HER2. HBP-FLAP binding to HER2-Fc, noncoated well (PBS), or EGFR-Fc were evaluated by ELISA. The mean 
SEM of three independent experiments is shown; *p < 0.05.

highest binding aﬃnity toward HER2 (KD ¼ 58 nM in ELISA andKD ¼ 287 nM in biolayer interferometry; Table 2 and Fig. S1B†)
and was selected for further characterization.

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3.4

Characterization of HBP-FLAP in vitro

The proteolysis-resistance of p1.M.2 peptide (YCAHNM) was
evaluated by treatment with proteinase K, which cleaves peptide
bonds adjacent to the carboxyl group of the 1st (Y) and 3rd (A)residues (Fig. 4A). A C-terminally elongated synthetic p1.M.2
peptide (YCAHNM-GSGSGK) was used for easy detection of
peptide-proteolysis by HPLC. As expected, the p1.M.2 peptide in
FN3 was resistant to proteinase K treatment for 2 h, whereas thelinear p1.M.2 was digested within 10 min (Fig. 4B and S2†).
The binding specicity of FN3-p1.M.2, hereinaer referred toas HBP-FLAP, was then evaluated using cell lines expressing
diﬀerent levels of HER2. HeLa cells (no HER2 expression), SHSY5Y cells (no HER2 expression), SK-BR-3 cells (HER2 expression), or N87 cells (HER2 expression) were incubated with the
HBP-FLAP, followed by staining with a uorescently labeledanti-His-tag antibody that binds to the His-tag at the N-terminusof FN3. The uorescence intensity of the cells was observed tocorrelate with the expression level of HER2 (Fig. 4C and D). The
specicity of HBP-FLAP for HER2-Fc was further conrmed by
an ELISA using epidermal growth factor receptor (EGFR)-Fccoated wells: HBP-FLAP hardly bound to EGFR-Fc-coated wells(Fig. 4E). These results showed that the HBP-FLAP has highspecicity for HER2.

Paper
3.5 In vivo uorescent imaging of a HER2-expressing tumor
by HBP-FLAP
To assess the applicability of HBP-FLAP as a probe for targeting a HER2-expressing tumor in vivo, the HBP-FLAP and
FN3 scaﬀold were labeled with a near-infrared uorescent dye
IR800CW (HBP-FLAP-IR800 and FN3-IR800; MW ¼ 10 kDa)
(Fig. 1D, 5A and S3†). In vivo imaging was performed atvarious post-injection (p.i.) times to monitor HBP-FLAP-IR800
and FN3-IR800 probes in N87 tumor-bearing nude mice.
Although similar uorescent intensities were observed intumors at 0.5 h p.i. of the probes, higher uorescent signals
were detected at longer periods in tumors from mice injectedwith HBP-FLAP-IR800 (Fig. 5B and C). HBP-FLAP-IR800
specically accumulated at HER2-expressing tumors withhigher T/B ratios when compared with that of FN3-IR800(Fig. 5D). As expected from the molecular weight of theprobes, both probes were mainly excreted through the urinary
system (Fig. 5B). These results suggested that binding to
HER2 retained HBP-FLAP for longer periods in tumors andthat the HBP-FLAP design is a promising strategy for developing clinically applicable small proteins using targetbinding peptides.

Fig. 5 In vivo ﬂuorescence imaging of a HER2-expressing tumor. (A) Average ﬂuorescent intensity (photons per s per cm2 per sr) of HBP-FLAPIR800 and FN3-IR800 probe before i.v. injection; n ¼ 3, mean  SEM. (B) Representative in vivo images of N87 tumor-bearing mice at theindicated times of p.i. of the probes shown in (A). A dashed blue circle indicates the tumor, a black arrow indicates the liver, an orange arrow
indicates the kidney and the blue arrow indicates the bladder. (C) Average ﬂuorescent intensity (photons per s per cm2 per sr) of HBP-FLAP-IR800
and FN3-IR800 probes in tumors. (D) The ratio of tumor (ﬂuorescence intensity of tumors) versus background (ﬂuorescence intensity of muscles)(T/B ratio) of HBP-FLAP-IR800 and FN3-IR800 probes at the indicated times of p.i.

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4. Discussion

5.

In this study, we successfully developed an HBP-FLAP thatspecically binds to HER2 (Table 2 and Fig. 4D) and showed
applicability for targeting HER2-expressing tumors in vivo (Fig. 5).
Although target-binding peptides/proteins selected by in vitro
screening may bind to oﬀ-targets in vivo, HBP-FLAP likely binds to
HER2 specically because no detectable signal was observed inparts of the body other than HER2-expressing tumors and excretion organs (Fig. 5B). In the evaluation of FN3-HBPs (Fig. 2D and3A), all graed hexapeptides were immobilized into the FN3
scaﬀold and three FN3-HBPs bound to HER2, whereas the other
FN3-HBPs may lose critical residues for binding. This result showsthat the design method of FLAP is a promising strategy to develop
target-binding proteins by graing target-binding peptides to theGA site of the FN3 scaﬀold and can contribute to the developmentof clinically applicable target-binding proteins. In comparison to
HBP, HBP-FLAP was more resistant to proteinase K (Fig. 4B). The
HBP of HBP-FLAP must be exposed on the surface of the molecule
because the GA site was selected because it is a solvent exposed
loop in the FN3 structure.18 Therefore, the proteolysis resistance ofthe HBP in HBP-FLAP is not because of reduced protease access tothe target sequence but is likely due to its immobilized structure,
which restricts protease activity.
The linear HER2-targeting peptide (KCCYSL)9 and cyclic
peptide (FCGDGFYACYMDV)11 reported in other studies
showed less serum stability, which might be caused bysusceptibility to proteases. These peptides were PEGylated toincrease resistance to proteases and the molecular mass,
resulting in prolonged blood circulation.26,27 Another study
demonstrated that conjugation of a bicyclic peptide to analbumin-binding peptide increased proteolysis-resistance.28
Our study reported a diﬀerent strategy to improve theproteolysis-resistance of HBP by peptide graing, which directly
immobilized the conformation of the peptide.
The FLAP design might be a preferable strategy for developing clinically applicable proteolysis-resistant molecules fromtarget-binding peptides when compared with cyclization or
graing into cyclic peptide approaches, because many proteins
with a variety of molecular sizes and charges can be used as
scaﬀolds for developing FLAPs. Therefore, we can optimize thein vivo retention time of FLAPs by using the appropriate scaffolds. In comparison to other strategies such as PEGylation, Fcfusion and albumin-fusion, in which either the C- or Nterminus of the target-binding peptides is exposed, the FLAP
design can mask both termini by covalent conjugation with the
scaﬀold, suggesting that peptides in FLAP also show higher
proteolysis-resistance to exo-proteases in the body whencompared with that of peptides with other modications.
We showed the application of HBP-FLAP as a diagnostic
probe (in vitro immunostaining and in vivo imaging) in thisstudy. To address the applicability of HBP-FLAP for tumor
treatment, we need to further evaluate inhibition of HER2signaling, cancer cell proliferation and tumor growth. Theragnostic probes by radioisotope- and near-infrared dyeconjugations are also possible applications of FLAPs.

We successfully developed a HER2-binding protein, HBP-FLAP,by applying the design method of FLAP. HBP-FLAP specicallybinds to HER2 and has improved resistance to proteases. In
addition, HBP-FLAP exhibited in vivo applicability as a HER2expressing tumor-targeting probe. FLAP design can be applied
to any other target-binding peptides. Thus this study provides
a promising strategy for creating useful tools for basic,
preclinical and clinical studies.

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Conclusion

Author contributions
S. K.-K. and T. K. designed and supervised the experiments. Y.
O., T. K., M. K., T. S., M. T. and Y. I. provided T7 phage libraries
and performed screenings. W. Y., Y. O., T. K., S. S., H. N. and T.
Ku. validated the designed proteins. W. Y., T. K. and S. K.-K.
wrote the manuscript.

Conﬂicts of interest
There are no conicts to declare.

Acknowledgements
We are thankful to Biomaterials Analysis Division, Technical
Department, Tokyo Institute of Technology for their DNA
sequence analyses. We also sincerely thank Dr Hiroshi Ueda,
Institute of Innovative Research, Tokyo Institute of Technology
for the support of biolayer interferometry. This research waspartially supported by the JSPS KAKENHI, Grant Number
JP19K06970 (to T. K.). We thank the Edanz Group (https://enauthor-services.edanzgroup.com/) for editing a dra of thismanuscript.

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