pharmaceutics
ArticleAntitumor Activity of an Anti-EGFR/HER2 Bispecific Antibody
in a Mouse Xenograft Model of Canine Osteosarcoma
Nami Tateyama 1,† , Hiroyuki Suzuki 2, *,† , Tomokazu Ohishi 3,4,† , Teizo Asano 1 , Tomohiro Tanaka 1 ,Takuya Mizuno 5 , Takeo Yoshikawa 6 , Manabu Kawada 4 , Mika K. Kaneko 1 and Yukinari Kato 1,2,6, *
1

2

3

4

5

6

*
†

Citation: Tateyama, N.; Suzuki, H.;
Ohishi, T.; Asano, T.; Tanaka, T.;
Mizuno, T.; Yoshikawa, T.; Kawada,
M.; Kaneko, M.K.; Kato, Y. Antitumor
Activity of an Anti-EGFR/HER2
Bispecific Antibody in a Mouse
Xenograft Model of Canine
Osteosarcoma. Pharmaceutics 2022, 14,2494. https://doi.org/10.3390/
pharmaceutics14112494
Academic Editor: Isabelle Turbica
Received: 28 September 2022
Accepted: 15 November 2022
Published: 17 November 2022
Publisher’s Note: MDPI stays neutral

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Miyagi, Japan
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
18-24 Miyamoto, Numazu-shi 410-0301, Shizuoka, Japan
Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology, Microbial Chemistry Research Foundation,
3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary Medicine,
Yamaguchi University, 1677-1, Yoshida 753-8515, Yamaguchi, Japan
Department of Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku,
Sendai 980-8575, Miyagi, Japan
Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-22-717-8207 (H.S. & Y.K.)
These authors contributed equally to this work.

Abstract: The overexpression of epidermal growth factor receptors (EGFRs) has been reported invarious human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanizedanti-EGFR and anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs)
have been shown to improve patients’ survival. Canine tumors resemble human tumors in theinitiation and progression. We previously established a defucosylated mouse-dog chimeric antiEGFR mAb (E134Bf) and a mouse-dog chimeric anti-HER2 mAb (H77Bf), which exerted antitumor
activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf
single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognizeddog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow
cytometry. E134Bf-H77scFv also reacted with dEGFR/dHER2-positive canine osteosarcoma D-17
cells, and possesses a high binding-affinity (KD : 1.3 × 10−9 M). Furthermore, E134Bf-H77scFv exerted
antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells
in the presence of canine mononuclear cells and complement, respectively. Moreover, administrationof E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early comparedwith the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv
exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable
treatment regimen for canine tumors.

with regard to jurisdictional claims inpublished maps and institutional affil-

Keywords: EGFR; HER2; bispecific antibody; ADCC; CDC; canine osteosarcoma

iations.

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Therapeutic monoclonal antibodies (mAbs) for tumors have revolutionized over
several decades due to the development of the hybridoma technique [1] and the mAbhumanization technique [2]. Currently, more than 100 mAbs have been approved by the
US Food and Drug Administration (FDA) for the treatment of not only tumors, but also
inflammatory diseases [3]. MAbs can bind to target antigens with high specificity, andinhibit tumor promoting growth factor signaling pathways through their neutralization
effects. In addition, mAbs potentiate the host immune responses including antibody

Pharmaceutics 2022, 14, 2494. https://doi.org/10.3390/pharmaceutics14112494

https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2022, 14, 2494

2 of 14dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), andantibody-dependent cellular phagocytosis (ADCP) [4]. Due to the successful applicationof immunoglobulin G (IgG) type mAbs, several antibody formats including bispecificantibodies and antibody–drug conjugates (ADCs) have been approved by the FDA as next
generation therapeutic agents for various types of tumors [5,6].
Targeting receptor tyrosine kinase signaling pathways is one of the important strategies for tumor therapy [7]. Human epidermal growth factor receptor (HER) families belong
to type I transmembrane glycoproteins that are overexpressed in various solid tumors [8].
Through their homo- and hetero-dimerization, a broad repertoire of oncogenic signaling
pathways is activated, and these are essential for tumor development [9]. Anti-epidermal
growth factor receptor (EGFR) mAb, cetuximab, can block EGFR activation through thebinding to its ligand binding domain and, thus, inhibit the EGFR signaling pathways.
Moreover, cetuximab (IgG1 isotype) exhibits ADCC activity, and directs cytotoxic immune
effector cells to EGFR-positive tumor cells [10,11]. Cetuximab was approved in combination
with platinum-based chemotherapy, showing good outcomes including overall survival, response rate, and progression-free survival in metastatic/recurrent head and neck squamouscell carcinomas [12]. Trastuzumab, the first FDA-approved anti-HER2 mAb, has been themost effective therapy for HER2-positive breast cancers and later in HER2-positive gastriccancers for more than 20 years [13]. Clinically, the efficacy is mediated by the involvementof immunologic engagements, including ADCC, CDC, and ADCP activity [4].
With the increase in lifespan of both humans and dogs, the tumor incidence has
increased, as well. Spontaneous canine tumors closely look like human tumors [14]. Osteosarcoma is the most common primary bone tumor in dogs. The rate of osteosarcoma indogs is 27 times higher than that in humans, and lung metastasis is frequently observed. In
fact, a one-year survival rate is only 45% [15]. Currently, treatment options for osteosarcoma in dogs include surgery, radiation, and chemotherapy [16,17]. However, sufficienttherapeutic efficacies have not been reported. Therefore, the development of other therapeutic options for canine osteosarcoma is necessary. Specifically, EGFR expression was
significantly upregulated in osteosarcoma lung metastasis compared to the extrapulmonary
sites [18]. Doyle et al. reported the vaccine therapy using dog EGFR (dEGFR)/dog HER2(dHER2) peptide in canine osteosarcoma. Dogs with osteosarcoma were immunized witha dEGFR/dHER2 peptide vaccine, which resulted in an increased anti-dEGFR/dHER2
antibody production and T cells infiltration into tumors. The vaccinated dogs with osteosarcoma showed tumor regression and increased survival. These data support the approachof amplifying antitumor immunity by dEGFR/dHER2 peptide [19].
The ADCC is activated by the binding of the FcγRIIIa on natural killer (NK) cells to
the Fc region of mAbs [4]. However, the N-linked glycosylation in the Fc region is reported
to reduce the binding to the FcγRIIIa on NK cells [20,21]. Fucosyltransferase 8 (FUT8) is
an important enzyme that mediates the N-linked glycosylation (core fucosylation) in the
Fc region [21]. Therefore, FUT8-knockout Chinese hamster ovary (CHO) cells have beenshown to produce completely defucosylated recombinant antibodies. The defucosylated
mAb strongly binds to FcγRIIIa and exerts potent ADCC activity when compared to aconventional mAb [22].
Previously, we developed an anti-EGFR mAb (EMab-134) [23] and anti-HER2 mAb(H2 Mab-77) [24] using the Cell-Based Immunization and Screening method. Subsequently,
we produced mouse-dog chimeric anti-EGFR mAb (E134B) and anti-HER2 mAb (H77B)
from the information of variable regions of EMab-134 and H2 Mab-77, respectively. Furthermore, we evaluated the antitumor effect of their respective defucosylated forms,
E134Bf [25,26] and H77Bf [27] against canine tumors in mouse xenograft models. In
this study, we produced a defucosylated bispecific Ab against dEGFR and dHER2, andevaluated the ADCC, CDC, and antitumor activities in canine osteosarcoma D-17 xenograft.

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2. Materials and Methods
2.1. Cell Lines
CHO-K1 and a canine osteosarcoma cell line D-17 were purchased from the American
Type Culture Collection (Manassas, VA, USA). Stable dog EGFR-overexpressed CHOK1 (CHO/dEGFR) [26] and dog HER2-overexpressed CHO-K1 (CHO/dHER2) [27] were
established as described previously. CHO-K1, CHO/dEGFR, and CHO/dHER2 were
cultured in RPMI-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), containing 10%
fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 µg/mL
streptomycin, 100 units/mL of penicillin, and 0.25 µg/mL amphotericin B (Nacalai Tesque,Inc.). D-17 was cultured in MEM medium (Nacalai Tesque, Inc.), supplemented with 10%
FBS, 1 mM of sodium pyruvate, 100 units/mL of penicillin, 100 µg/mL streptomycin, and0.25 µg/mL amphotericin B.
2.2. Animals
In all animal experiments, we followed the guidelines of the Institutional Committee
for Experiments of the Institute of Microbial Chemistry (Numazu, Japan) (approval no.
2022-036). Mice were bred in a specific pathogen-free condition on an 11 h light/13 h darkcycle. Mice weight were monitored two times per week and health were monitored threetimes per week. The loss of original body weight was determined to a point >25% and/or amaximum tumor size >2000 mm3 and/or significant changes in the appearance of tumors
as humane endpoints for euthanasia. Cervical dislocation was used for euthanasia. Micedeath was confirmed by respiratory arrest and rigor mortis.
2.3. Antibodies
Previously, we have established mouse-dog chimeric anti-EGFR mAb (E134B) [26] andanti-HER2 mAb (H77B) [27]. In this study, to generate E134Bf-H77scFv, we first constructeda single chain Fv of H77B (H77scFv) by connecting the VH and VL cDNA of H77B with alinker sequence (GGGGSGGGGSGGGGS). The H77scFv cDNA was further fused at the
30 end of the light chain cDNA of E134B. The cDNA of the E134B heavy chain and E134B
light chain-H77scFv were transduced into BINDS-09 (FUT8 knockout ExpiCHO-S) cells
using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.) to produce thedefucosylated form [25–32]. A defucosylated and bispecific antibody, E134Bf-H77scFv, waspurified using Ab-Capcher (ProteNova Co., Ltd., Kagawa, Japan). We confirmed theirpurity by SDS-PAGE (Supplemental Figure S1). Dog IgG was purchased from Jackson
ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).
2.4. Flow Cytometry
CHO-K1, CHO/dEGFR, CHO/dHER2, and D-17 cells were suspended in blocking buffer [0.1% bovine serum albumin in phosphate-buffered saline (PBS)] containing
1 µg/mL of E134Bf, H77Bf, E134Bf-H77scFv for 30 min at 4 ◦ C. Then, cells were suspendedin blocking buffer containing Alexa Fluor 488-conjugated anti-dog IgG (1:1000; Jackson
ImmunoResearch Laboratories, Inc.) for 30 min at 4 ◦ C. Fluorescence data were collectedby the Cell Analyzer EC800, and analyzed by EC800 software ver. 1.3.6 (Sony Corp.,
Tokyo, Japan).
2.5. Determination of Binding Affinity
Serially diluted E134Bf (0.0006–10 µg/mL), H77Bf (0.0006–10 µg/mL), and E134BfH77scFv (0.0006–10 µg/mL) were suspended with CHO/dEGFR, CHO/dHER2, and D-17
cells. The cells were further treated with Alexa Fluor 488-conjugated anti-dog IgG (1:100).
Fluorescence data were obtained by the Cell Analyzer EC800. To determine the apparent
dissociation constant (KD ), GraphPad Prism version 8 (the fitting binding isotherms tobuilt-in one-site binding models, GraphPad Software, Inc., La Jolla, CA, USA) was used.

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4 of 142.6. ADCC
Induction of ADCC by E134Bf, H77Bf, and E134Bf-H77scFv was assayed as follows.
Canine mononuclear cells (MNCs, from Yamaguchi University) were resuspended in
DMEM supplemented with 10% FBS, and were used as effector cells. Target D-17 cells werelabeled with 10 µg/mL of Calcein AM (Thermo Fisher Scientific, Inc.) [25–42]. The effector
canine MNCs and the target cells (effector/target cells ratio, 50) were incubated in thepresence of 100 µg/mL of control dog IgG, E134Bf, H77Bf, and E134Bf-H77scFv for 4.5 h at
37 ◦ C. The Calcein release into the medium was measured with an excitation wavelength(485 nm) and an emission wavelength (538 nm) using a microplate reader (Power Scan HT;
BioTek Instruments, Inc., Winooski, VT, USA). Cytolyticity (% lysis) was determined as
described previously [27].
2.7. CDC
The target D-17 cells were incubated with 10 µg/mL calcein AM [25–42], and wereincubated with rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada;
final dilution 1:10) in the presence of 100 µg/mL of control dog IgG, E134Bf, H77Bf, andE134Bf-H77scFv. The Calcein release into the medium was determined as indicated in theprevious section. Cytolyticity (% lysis) was determined as described previously [27].
2.8. Antitumor Activities in Xenografts of D-17
BALB/c nude mice (female, 4 weeks old) were purchased from Charles River Laboratories, Inc. D-17 cells (5 × 106 cells) were subcutaneously inoculated into the left flank ofmice together with BD Matrigel Matrix Growth Factor Reduced (BD Biosciences, San Jose,
CA, USA). On day 8 after the inoculation, 100 µg of E134Bf, H77Bf, and E134Bf-H77scFv,
and control dog IgG (n = 8) in PBS (100 µL) were intraperitoneally injected. Additional
antibodies were intraperitoneally injected on days 15 and 22. Furthermore, on days 8, 15,
and 22, canine MNCs (5 × 105 cells) were injected into the surrounding tumors. The tumor
volume was measured on days 8, 10, 15, 17, 22, and 25, as described previously [25–43].
3. Results
3.1. Flow Cytometric Analysis against dEGFR and dHER2-Expressing Cells Using E134Bf-H77scFv
We previously developed mouse-dog chimeric mAbs including E134B (anti-dEGFR)
and H77B (anti-dHER2), both of which possess the B type dog IgG backbone to confer the
ADCC activity. We further produced the corresponding defucosylated forms, E134Bf [26]and H77Bf [27], respectively. To generate a bispecific Ab against dEGFR and dHER2, we first
constructed the cDNA of H77scFv, following the ligation to the light chain cDNA of E134B(Figure 1). Subsequently, we transduced the cDNAs of E134B heavy chain and the E134B
light chain-H77scFv into FUT8 knockout ExpiCHO-S cells, and purified the defucosylated
bispecific Ab against dEGFR and dHER2, which is labeled as E134Bf-H77scFv.
We first confirmed the reactivity of E134Bf-H77scFv to dEGFR and dHER2 expressed
cells using flow cytometry. As shown in Figure 2, E134Bf-H77scFv exhibited the similar
reactivities to CHO/dEGFR (Figure 2A) and CHO/dHER2 (Figure 2B) cells compared with
E134Bf and H77Bf, respectively. In contrast, E134Bf-H77scFv never reacted with parental
CHO-K1 cells (Figure 2C). Thereafter, we examined the reactivity of E134Bf-H77scFv against
dEGFR and dHER2-positive canine osteosarcoma D-17 cells. As shown in Figure 2D, E134BfH77scFv could recognize D-17 cells. Then, we determined apparent dissociation constants(KD ) of E134Bf, H77Bf and E134Bf-H77scFv to D-17 cells using flow cytometry. As shownin Figure 3, the KD values for the interaction of E134Bf, H77Bf, and E134Bf-H77scFv with
D-17 cells were 6.0 × 10− 10 M, 2.9 × 10− 10 M, and 1.3 × 10− 9 M, respectively.

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5 of 14

Figure 1.1.Schematic
illustrationof theof
productionof bispecific
E134Bf-H77scFv
from E134B
FigureSchematic
illustration
the productionof antibody,bispecificantibody, E134Bf-H77scFv
fro
and
H77B.
E134B and H77B.

We first confirmed the reactivity of E134Bf-H77scFv to dEGFR and dHER2 expresse
cells using flow cytometry. As shown in Figure 2, E134Bf-H77scFv exhibited the simila
reactivities to CHO/dEGFR (Figure 2A) and CHO/dHER2 (Figure 2B) cells compared wit
E134Bf and H77Bf, respectively. In contrast, E134Bf-H77scFv never reacted with parent
CHO-K1 cells (Figure 2C). Thereafter, we examined the reactivity of E134Bf-H77scF
against dEGFR and dHER2-positive canine osteosarcoma D-17 cells. As shown in Figur
2D, E134Bf-H77scFv could recognize D-17 cells. Then, we determined apparent dissociation constants (KD) of E134Bf, H77Bf and E134Bf-H77scFv to D-17 cells using flow cytometry. As shown in Figure 3, the KD values for the interaction of E134Bf, H77Bf, and E134B

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Figure 2. Flow cytometry using E134Bf, H77Bf, and E134Bf-H77scFv. CHO/dEGFR (A), CHO/dHER2(B), CHO-K1 (C), and D-17 (D) cells were treated with 1 µg/mL of E134Bf, H77Bf, and E134Bf-H77scFv
or buffer control, followed by Alexa Fluor 488-conjugated anti-dog IgG. Fluorescence data wereanalyzed using the Cell Analyzer EC800.

Figure 2. Flow cytometry using E134Bf, H77Bf, and E134Bf-H77scFv. CHO/dEGFR
CHO/dHER2 (B), CHO-K1 (C), and D-17 (D) cells were treated with 1 μg/mL of E134Bf, H77Bf,
E134Bf-H77scFv or buffer control, followed by Alexa Fluor 488-conjugated anti-dog IgG. Fluo
cence data were analyzed using the Cell Analyzer EC800.

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7 of7 15
of 14

Figuredeterminationof theapparent
dissociation
constantof E134Bf,
H77Bf,
E134BfFigure
3. 3.
TheThedeterminationof theapparent
dissociation
constantof E134Bf,
H77Bf,
andandE134BfH77scFv
D-17
cells
using
flow
cytometry.
D-17
cells
were
incubatedwithserially
diluted
E134Bf,
H77scFv
forfor
D-17
cells
using
flow
cytometry.
D-17
cells
were
incubatedwithserially
diluted
E134Bf,
H77Bf,
andE134Bf-H77scFv,
followed
treatment
with
Alexa
Fluor
488-conjugatedanti-dog
H77Bf,
andE134Bf-H77scFv,
followed
byby
thethetreatment
with
Alexa
Fluor
488-conjugatedanti-dog
IgG.
Fluorescence
data
were
following thethecalculation
calculationofofthe
IgG.
Fluorescencedata
wereanalyzed
analyzedusing
usingthetheCell
Cell Analyzer
Analyzer EC800, following
theapparent
apparentdissociation
dissociationconstantconstant(K(KD)
byGraphPad
Prism
8.
)
by
GraphPad
Prism
8.
D

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8 of 15
8 of 143.2.
3.2.E134Bf-H77scFv-Mediated
E134Bf-H77scFv-MediatedADCC
ADCCand
andCDC
CDCininD-17
D-17Cells
Cells
We
next
investigatedwhether
E134Bf-H77scFv
wasWe next investigated whether E134Bf-H77scFv wascapable
capableofofmediating
mediatingADCC
ADCC
againstD-17
cells.
E134Bf-H77scFv
showedADCC(15.2%
cytotoxicity)
againstagainst D-17 cells. E134Bf-H77scFv showed ADCC (15.2% cytotoxicity) againstD-17
D-17cells
cells
more
thethecontroldog
IgGIgG(4.1(4.1%% cytotoxicity;p < p0.01).
ThereThere
was nosigmoreeffectively
effectivelythan
thancontroldog
cytotoxicity;< 0.01).
was
nonificant
difference
between
E134Bfor H77Bf
and thedog IgG
D-17 D-17
in thisinsignificant
difference
between
E134Bfor H77Bf
andcontrol
the control
dogagainst
IgG against
experimental
condition(Figure4A).4A).
We We
next
examined
whether
E134Bf-H77scFv
this experimental
condition(Figurenext
examined
whether
E134Bf-H77scFvcould
could
exert
exertCDC
CDCagainst
againstD-17
D-17cells.
cells.As
AsshownshownininFigure
Figure4B,
4B,H77Bf,
H77Bf,E134Bf,
E134Bf,and
andE134Bf-H77scFv
E134Bf-H77scFv
inducedinduceda ahigher
higherdegree
degreeofofCDC
CDC(33.6%(33.6%[p[p<<0.05],
0.05],40.7%
40.7%[p[p<<0.01],
0.01],andand44.4%
44.4%[p[p<<0.01]
0.01]
cytotoxicity,respectively)
in
D-17
cells
comparedwiththat
inducedbythecontroldog
cytotoxicity, respectively) D-17 cells compared with that induced by the control dog IgG
IgG(13.8%
cytotoxicity).
These
results
indicatedthat E134Bf-H77scFv
exhibitedhigherof(13.8%
cytotoxicity).
These
results
indicatedthat E134Bf-H77scFv
exhibitedhigher levels
levelsof andADCC
and
CDC activities
againstD-17 cells.
ADCC
CDC
activities
against D-17
cells.

Figure4.4.Evaluation
EvaluationofofADCC
ADCCandand
CDC
inducedbyby
E134Bf,
H77Bf,andand
E134Bf-H77scFv.(A)(A)ADCC
ADCC
FigureCDC
induced
E134Bf,
H77Bf,
E134Bf-H77scFv.
by
E134Bf,
H77Bf,
E134Bf-H77scFv,
andcontroldog
IgG
against
D-17
cells
in
the
presenceofcanineby E134Bf, H77Bf, E134Bf-H77scFv, and control dog IgG against D-17 cells in the presence of canine
MNCs.
(B)
CDC
elicited
by
E134Bf,
H77Bf,
E134Bf-H77scFv,
andcontroldog
IgG
targetingD-17
cells.
MNCs. (B) CDC elicited
E134Bf, H77Bf, E134Bf-H77scFv, and control dog IgG targeting D-17
cells.
Valuesarepresented
as
the
mean
±
SEM.
(**
p
<
0.01
and
*
p
<
0.05;
Welch’sttest).
ADCC,
Values are presented as the mean ± SEM. (** p < 0.01 and * p < 0.05; Welch’s t test). ADCC, antibodyantibody-dependent
cytotoxicity;
CDC, complement-dependent
cytotoxicity;mondependent cellular cellularcytotoxicity;
CDC, complement-dependent
cytotoxicity;
MNCs,MNCs,
mononuclear
onuclearcells;n.s.,
notsignificant.
cells; n.s., not significant.

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9 of 143.3. Antitumor Effects of E134Bf-H77scFv in the Mouse Xenograft of D-17
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9 of Cells
15
To evaluate the antitumor activity against D-17 xenograft tumor, E134Bf-H77scFv,
E134Bf, H77Bf and control dog IgG were intraperitoneally injected into mice on days 8, 15,
3.3. Antitumorof E134Bf-H77scFv
in thecells.
MouseFurthermore,
Xenograft of D-17
and
22 afterEffects
the inoculationof D-17
onCells
days 8, 15, and 22, canine MNCs
To
evaluatetheantitumor
activity
againstD-17
xenografttumor,
were injected surrounding the tumors. On days 8, 10, 15, 17,E134Bf-H77scFv,
22, and 25 after the inoculation,
E134Bf, H77Bf and control dog IgG were intraperitoneally injected into mice on days 8,
the
tumor
volumewasdetermined.
The
E134Bf-H77scFv
administration
resulted in faster
15, and 22 after the inoculation of D-17 cells. Furthermore, on days 8, 15, and 22, canine
reductionoftumor
volume
on
days
10
(p
<
0.05),
15(p<0.05),
and17(p<
MNCs were injected surrounding the tumors. On days 8, 10, 15, 17, 22, and 25 after the 0.01) than that of
inoculation,
tumorandvolumewas determined.
The E134Bf-H77scFv
administration
re-and 25, significant
theE134Bf,theH77Bfcontroldog IgG (Figure
5A). However,on days 22
sultedinfasterreductionoftumor
volume
on
days
10
(p
<
0.05),
15(p<0.05),
and17(p
reduction of tumor was also observed in E134Bf and H77Bf treated <groups as well as0.01) than that of the E134Bf, H77Bf and control dog IgG (Figure 5A). However, on days
theE134Bf-H77scFv treated group compared with the controls (Figure 5A). As shown in
22 and 25, significant reduction of tumor was also observed in E134Bf and H77Bf treatedFigureweightof D-17 tumors
treatedwithwith
E134Bf-H77scFv,
E134Bf, and H77Bfgroups as5B,
welltheas the
E134Bf-H77scFv
treated groupcomparedthe controls (Figure
wassignificantly
lowerthat treatedwith control
dogE134Bf-H77scFv,
IgG (70%, 64%, 58% reduction,
5A). As
shown in Figure
5B, thanthe weightof D-17 tumors
treated with
E134Bf, and H77Bf
significantly
lower thantreatedwith control
IgGon
(70%,
respectively;
p <was0.01).
D-17 tumors
thatthat
wereresected
fromdogmice
day 25 are shown in
64%, 58%5C.
reduction,
respectively;< 0.01). D-17
that were in
resected
miceonFigureThe lossof body pweight
wastumors
not observed
each fromgroup(Figure5D). The miceday 25 are shown in Figure 5C. The loss of body weight was not observed in each group
on
day
25
are
shown
in
Figure5E.
(Figure 5D). The mice on day 25 are shown in Figure 5E.

Figure 5.5.
Antitumor
activityof E134Bf,
H77Bf, andE134Bf-H77scFv.
(A) Evaluationof Evaluation
tumor vol- of tumor volumes
FigureAntitumor
activityof E134Bf,
H77Bf,and E134Bf-H77scFv.
(A)umes in D-17 xenograft. D-17 cells (5 × 106 cells) were subcutaneously inoculated into mice. On day
6 cells) were subcutaneously inoculated into mice. On day 8
in
D-17
(5 ×
8 afterthexenograft.
inoculation ofD-17
D-17 cells
cells, 100
g 10
of E134Bf,
H77Bf, E134Bf-H77scFv or control dog IgG
were
injected
intraperitoneally.
Additional
antibodies
were injected
onE134Bf-H77scFv
days 15 and 22. Canine
after the inoculation of D-17 cells, 100 µg of E134Bf,
H77Bf,or control dog IgG were
MNCs were also injected surrounding tumors on days 8, 15, and 22. The tumor volume wasinjected intraperitoneally. Additional antibodies were injected on days 15 and 22. Canine MNCs werealso injected surrounding tumors on days 8, 15, and 22. The tumor volume was determined on days
8, 10, 15, 17, 22, and 25. Values are presented as the mean ± SEM. ** p < 0.01 and * p < 0.05 (ANOVA
and Sidak’s multiple comparisons test). (B) Xenograft weight (day 25) was measured. Values arepresented as the mean ± SEM. ** p < 0.01 (Welch’s t test). (C) Resected xenografts appearance offrom the indicated groups on day 25 (scale bar, 1 cm). (D) Mice body weights inoculated with D-17
xenografts on days 8, 10, 15, 17, 22, and 25 (ANOVA and Sidak’s multiple comparisons test). n.s., notsignificant. (E) Body appearance of D-17- inoculated mice on day 25 (scale bar, 1 cm).

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4. Discussion
In this study, we developed a novel bispecific antibody E134Bf-H77scFv against dEGFR
and dHER2 (Figure 1), and showed more potent ADCC activity compared with that of
E134Bf and H77Bf (Figure 4A). Furthermore, E134Bf-H77scFv showed the antitumor activity
against D-17 xenografts at earlier periods compared with E134Bf and H77Bf treatments(Figure 5A), suggesting that E134Bf-H77scFv possesses the different mode of actions. As
shown in Figure 2, E134Bf-H77scFv recognized dEGFR and dHER2-overexpressed CHO-K1
cells. Furthermore, E134Bf-H77scFv also reacted with dEGFR and dHER2 double positive
D-17 cells. However, we have not determined whether E134Bf-H77scFv can induce thecross-link between dEGFR and dHER2 on the cell surface. In our epitope mapping analysis,
EMab-134, the original mAb of E134B, recognized the sequence (377 -RGDSFTHTPP-386 ) in
domain III of EGFR [44]. The EGFR peptide (amino acids 375-394) containing the epitope
potently inhibited the recognition of D-17 cells by E134Bf. In the same condition, theinhibition to E134Bf-H77scFv was weaker than that to E134Bf (Supplemental Figure S2),
suggesting that E134Bf-H77scFv reacts with dHER2 on D-17 cells. However, the epitope of
H77scFv has not been identified. Further investigations are essential to reveal the structural
relevance of recognition, and the mechanism of the antitumor activity.
E134Bf-H77scFv is a novel modality for targeting dEGFR and dHER2. Because
H77scFv are fused to the light chain of E134B but not to the heavy chain, E134Bf-H77scFv
recognizes dual antigens without affecting the Fc-mediated ADCC activity. Furthermore,in case of bispecific antibody with two unique Fab domains, the combination of VH and VL
cDNAs should be considered, and the selection of objective bispecific antibody is required.
In contrast, E134Bf-H77scFv can be produced by transfection with single combination of
VH and VL cDNA, and purified by the conventional Fc binding beads.
Previously, different types of dual-targeting EGFR and HER2 bispecific modalities
have been tested in preclinical studies. A bispecific affibody molecule to EGFR or HER2
was generated by linking a bivalent HER2-binding affibody to a bivalent EGFR-binding
affibody with a linker sequence (Gly4-Ser)3. The bispecific affibody was shown to bind toboth HER2-overexpressed SKBR-3 and EGFR-overexpressed A-431 cells [45]. “AffiMabs”
were generated by fusing an EGFR-targeting affibody molecule to trastuzumab’s heavy or
light chains. AffiMabs were shown to induce the downregulation of both EGFR and HER2.
Furthermore, AffiMabs exhibited a more potent anti-proliferative effect than trastuzumabin vitro [46]. Moreover, a bispecific nanobody targeting EGFR and HER2 (bsNb) was
developed. The bsNb was further ligated to rhamnose (Rha) hapten to reconstitute the Fc
effector biological function. The bsNb-Rha retained dual-targeting activity to EGFR and
HER2, and elicited potent antitumor effects in vivo via the Fc-mediated engagement ofendogenous anti-Rha antibodies [47]. However, bispecific antibody formats against EGFR
and HER2 have not been applied in the clinic at present. Studies have demonstrated thatoverexpression of EGFR family proteins is observed in aggressive canine tumors including
osteosarcoma [48] and hemangiosarcoma [49] with shortened the survival. We have alreadyshowed that EMab-134 and H2 Mab-77, original antibodies of E134Bf-H77scFv, can be usedin immunohistochemistry [23,24]. Therefore, EMab-134, H2 Mab-77, and E134Bf-H77scFv
are expected to be used in clinics for both the diagnosis and treatment of canine tumors.
Bispecific antibodies have been approved as therapeutic agents to cover unmet clinicalneeds [50,51]. A wide range of bispecific antibody formats has been developed for cancer
therapy through different mechanisms including the engagement of T cells or other immune
cells (e.g., NK cells) to induce antitumor immunity, and bridging receptors to inhibit or activate the downstream signaling pathways [52]. For targeting receptor kinase signaling pathways including EGFR, HER2, HER3, and MET in tumor cells, GBR1302 has been developed
as a T-cell engager to direct CD3-positive T cells to HER2 on tumor cells [53]. Furthermore,bispecific antibodies that recognize different receptors involved in signaling crosstalk canbe used to avoid bypass signal transduction during tumor development [54–56]. Growing
number of evidence has indicated that MET signaling contributes to the resistance of EGFR
tyrosine kinase inhibitors in non-small cell lung cancers (NSCLC) [57–59]. Therefore, dual

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inhibition of aberrant oncogenic signaling has become a promising strategy. Amivantamab
is a dual-targeting EGFR and MET bispecific antibody that can inhibit the EGFR and MET
signaling pathways [56]. In various tumor models, amivantamab efficiently downregulates
the expression of EGFR and MET, and exhibited antitumor immunity with Fc-mediatedeffector interactions [60]. The FDA granted accelerated approval to JNJ-61186372 (Amivantamab) for the treatment for locally advanced or metastatic NSCLC. Another example is
MCLA-128, a bispecific antibody for HER2 and HER3. MCLA-128 can inhibit heregulin(a HER3 ligand)-mediated signaling of HER2/HER3, and suppress tumor cell survival
and proliferation via the downregulation of PI3K/Akt signaling [61]. Clinical studies on
MCLA-128 are ongoing in patients with breast cancer, pancreatic cancer, and NSCLC.
For the development of E134Bf-H77scFv to treat canine tumors in the clinic, the effectof E134Bf-H77scFv on dEGFR homodimer and dEGFR/dHER2 heterodimer-mediated signaling should be investigated. Further, it should be determined whether E134Bf-H77scFv
can induce the internalization of dEGFR and dHER2. Canine tumors represent an outbred
population, and resemble human tumors in the initiation, disease progression, growth factors and oncogene mutations. A growing number of evidences has suggested that elevatedexpression of other HER family and their ligands confer the resistance to trastuzumab [62].
For instance, co-expression of EGF in HER2-positive cell lines reduced the growth inhibitory
effect of trastuzumab by modulating EGFR/HER2 interaction [63]. Furthermore, EGFR
expression was associated with worse prognosis, specifically when co-expressed with HER2
in advanced breast cancer [64]. Therefore, future investigations could contribute to not
only canine, but also human EGFR/HER2-positive tumor treatment.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pharmaceutics14112494/s1, Figure S1. Purity of E134Bf-H77scFv.
Figure S2. Flow cytometry using the EGFR peptide containing E134Bf epitope.
Author Contributions: N.T., T.O., T.T., T.M., T.Y. and T.A. performed the experiments. M.K.K., M.K.
and Y.K. designed the experiments. N.T., T.O., H.S. and Y.K. analyzed the data. N.T., H.S. and Y.K.
wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: The present study was supported in part by the Japan Agency for Medical Research andDevelopment (AMED; grant nos: JP22ama121008, JP21am0401013, JP22bm1004001, JP22ck0106730
and JP21am0101078).
Institutional Review Board Statement: The animal experiments for antitumor activity were performed (approval no. 2022-036) by the Institutional Committee for Experiments of the Institute ofMicrobial Chemistry (Numazu, Japan).
Data Availability Statement: The data presented in this study are available in the article and supplementary material.
Acknowledgments: The authors would like to thank Saori Okuno, and Saori Handa (Departmentof Antibody Drug Development, Tohoku University Graduate School of Medicine) for technicalassistance of in vitro experiments, and Shun-ichi Ohba and Akiko Harakawa [Institute of Microbial Chemistry (BIKAKEN), Numazu, the Microbial Chemistry Research Foundation] for technicalassistance of animal experiments.
Conflicts of Interest: The authors declare no conflict of interest.

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