Article

Locally misfolded HER2 expressed on cancer cells is
a promising target for development of cancerspecific antibodies
Graphical abstract

Authors

Cancer cells

Normal cells

Takao Arimori, Emiko Mihara,
Hiroyuki Suzuki, ..., Mika K. Kaneko,
Junichi Takagi, Yukinari Kato

Correspondence
M611-D618

M611-D618

arimori@protein.osaka-u.ac.jp (T.A.),
yukinari.kato.e6@tohoku.ac.jp (Y.K.)

misfold

In brief
Arimori et al. established a cancerspecific antibody that binds to humanepidermal growth factor receptor 2(HER2) on cancer cells but not to HER2 on
normal cells. A crystal structure and cellbased binding analysis revealed that thisspecificity is due to the recognition of a
local misfolded region, which is unique to
HER2 on cancer cells.

HER2

VL
VH
cell surface

Cancer-specific Antibody

HighlightsdA cancer-specific anti-HER2 antibody, H2Mab-214, wasestablishedd

H2Mab-214 binds only to HER2 on cancer cells, but not to
HER2 on normal cellsdH2Mab-214 recognizes a locally misfolded region of HER2d

Locally misfolded cell surface proteins can be targets forcancer-specific antibodies

Arimori et al., 2024, Structure 32, 536–549
May 2, 2024 ª 2024 Elsevier Ltd.
https://doi.org/10.1016/j.str.2024.02.007ll

ll
ArticleLocally misfolded HER2 expressed on cancer cells
is a promising target for developmentof cancer-specific antibodies
Takao Arimori,1,6,* Emiko Mihara,1 Hiroyuki Suzuki,2,3 Tomokazu Ohishi,4,5 Tomohiro Tanaka,2 Mika K. Kaneko,2,3
Junichi Takagi,1 and Yukinari Kato2,3,*
1Institute for Protein Research, Osaka University, 3-2. Yamadaoka, Suita, Osaka 565-0871, Japan
2Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi

980-8575, Japan
3Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi
980-8575, Japan
4Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, 18-24, Miyamoto, Numazu, Shizuoka
410-0301, Japan
5Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology, Microbial Chemistry Research Foundation, 3-14-23, Kamiosaki,
Shinagawa-ku, Tokyo 141-0021, Japan
6Lead contact*Correspondence: arimori@protein.osaka-u.ac.jp (T.A.), yukinari.kato.e6@tohoku.ac.jp (Y.K.)
https://doi.org/10.1016/j.str.2024.02.007

SUMMARY

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is associated with a poor prognosis, making it an important therapeutic target. Here, we establish a novel cancerspecific anti-HER2 antibody, H2Mab-214. H2Mab-214 reacts with HER2 on cancer cells, but unlike the therapeutic antibody trastuzumab, it does not react with HER2 on normal cells in flow cytometry measurements.
A crystal structure suggests that H2Mab-214 recognizes a structurally disrupted region in the HER2 domain
IV, which normally forms a b-sheet. We show that this misfolding is inducible by site-directed mutagenesis
mimicking the disulfide bond defects that also may occur in cancer cells, indicating that the local misfoldingin the Cys-rich domain IV governs the cancer-specificity of H2Mab-214. Furthermore, we show that H2Mab214 effectively suppresses tumor growth in xenograft mouse models. Our findings offer a potential strategy
for developing cancer-specific therapeutic antibodies that target partially misfolded cell surface receptors.

INTRODUCTION
Human epidermal growth factor receptor (HER) family of receptor tyrosine kinases consists of four members: EGFR (HER1),
HER2, HER3, and HER4. They play important roles in regulatingcell proliferation, differentiation, and migration, while being
implicated in many cancers. More than ten ligands for HER members have been identified, including epidermal growth factor,
transforming growth factor a, and neuregulins (NRGs) 1–4, yet
HER2 has no known direct activating ligand.1 The extracellularregion of all HER family members is commonly composed offour domains (I–IV). In the absence of ligands, those receptors
other than HER2 are stabilized in an inactive ‘‘tethered’’ conformation where a protruding loop (also called dimerization arm) inthe domain II interacts with the domain IV.2–4 Upon ligand binding, the receptors undergo a large conformational change from‘‘tethered’’ to ‘‘extended’’ and form homo- or heterodimers via
the dimerization arm, resulting in the autophosphorylation of
the cytoplasmic tyrosine kinase domain.5–7 HER2 is unique inthat it always adopts the extended conformation even in the
536 Structure 32, 536–549, May 2, 2024 ª 2024 Elsevier Ltd.

monomeric state.8 It has recently been shown that the structureof HER2 forming a heterodimer with HER3 is very similar to thatof the monomeric state.9 Hence, HER2 is always ready to be dimerized at cell surface, making it a preferable dimerization
partner for other HER family members.10 In addition, overexpression of HER2 promotes the formation of HER2 homodimers,
leading to ligand-independent signaling.11 HER2 overexpression
is found in 20% of breast cancers and is associated with poor
prognosis, higher rates of recurrence, and shorter overall survival.12 HER2 overexpression is also observed in a variety ofother cancers, including gastric, bladder, ovarian, and non-small
cell lung cancers.13–16
Trastuzumab, a monoclonal antibody (mAb) against HER2,
recognizes the domain IV8 and exhibits an anti-proliferating
effect in vitro and a potent antitumor effect in vivo.17 The additionof trastuzumab treatment to a standard chemotherapy improves
objective response rates, progression-free survival, and overall
survival in HER2-positive breast cancer patients with metastasis.18 Trastuzumab has been the standard treatment for
HER2-positive breast cancers19 and HER2-positive gastricllArticlecancers.20 Clinically, the efficacy of trastuzumab involves immunologic engagement.17 The Fc domain of trastuzumab mediates
the engagement with Fcg receptors (FcgRs) on various immune
cells. Trastuzumab-FcgR binding allows for phagocytosis of antibody-bound tumor cells, which is called antibody-dependent
cellular phagocytosis. The FcgR engagement also activates
macrophages, dendritic cells, and neutrophils, which can change
adaptive immune responses through chemotaxis, cytokine
production, and antigen presentation. Furthermore, the FcgR
engagement mediates the activation of natural killer (NK) cells
which attack and lyse the target tumor cells, termed antibodydependent cellular cytotoxicity (ADCC).21 Margetuximab wasdeveloped by introducing several mutations in trastuzumab to
improve the FcgRIIIA engagement (and thus ADCC activity),22
and was approved by US Food and Drug Administration (FDA)
and showed significant improvement in progression-free survival
in heavily pretreated patients.23,24 In addition to ADCC, trastuzumab is known to affect HER2 downstream signaling. Many in vitro
experiments suggested that trastuzumab arrests the cell cycle
through inhibiting the phosphatidylinositol 3-kinase (PI3K)/
protein kinase B (AKT) signaling pathway, which is highly activated in a variety of cancers.25 The detailed mechanism remains
controversial, but this is recognized as one of the major mechanisms of action of trastuzumab, as is its ADCC activity.
Although trastuzumab exhibits potent antitumor effect, somepatients develop resistance against trastuzumab treatment,
which can be attributed to the hyperactivation caused by theheterodimerization with HER3.26 Pertuzumab, another clinicallyapproved HER2-targeting mAb, recognizes the domain II andprevents NRG1-induced heterodimerization with HER3 andintracellular signaling.27 Thus, pertuzumab is considered tohave a complementary mechanism of trastuzumab,28 and the
double anti-HER2 blockade has become the standard therapy
in the initial management of metastatic HER2-positive breast
cancer.18 Furthermore, combination therapy using trastuzumab,
pertuzumab, and chemotherapy has been evaluated and foundto show even higher clinical benefit.29
Despite the proven success of HER2-targeted immunotherapies described previously, several adverse effects have also
been reported, including cardiotoxicity.30 Although they are notconsidered a clinically significant problem, routine cardiac monitoring is required in clinic during the anti-HER2 antibody treatment.30 Interestingly, mice lacking ErbB2 (ortholog of HER2)
display embryonic lethality due to the dysfunctions associatedwith a lack of cardiac trabeculae,31 and ventricular-restricted
ErbB2 deficiency in mice shows features of dilated cardiomyopathy.32 These results indicate that HER2 plays important role(s) innormal heart development and homeostasis. As a proteomic
study of healthy human tissues revealed a high level of broad
expression of HER2,33 anti-HER2 mAb therapies may attack
normal tissues not limited to the heart, which could lead to otherside effects including ones that are not clearly recognized at present. Therefore, it is highly desirable to use anti-HER2 mAbs thatcan exclusively recognize HER2 expressed on cancer cells,
although such reagents have not been available to this day.
In this study, we succeeded in establishing a novel mAb that
preferentially binds to HER2 expressed on cancer cells but not
to HER2 expressed on normal cells. This antibody, H2Mab214, was established through standard immunization and hy-

bridoma technology, but incorporating a special screening strategy called cancer-specific mAb or CasMab method we havedeveloped previously.34–37 The cancer-specificity of H2Mab214 was broadly investigated by flow cytometry analysis using
various cell lines of normal or cancer origins, and its antitumor
potential was confirmed by in vivo xenograft model. Most importantly, we clarified the molecular basis of H2Mab-214’s cancer
selectivity by determining the crystal structure of its complex
with HER2-derived peptide. Combined with the structure-guided
mutagenesis at the epitope region, we show evidence that the
local misfolding of HER2 domain IV Cys-rich region governs
the cancer-specificity of H2Mab-214, pointing toward a new
way of searching cancer-specific antibodies against various
cell surface receptors.
RESULTS
Cancer-specific HER2 recognition by H2Mab-214
We have previously established a mouse anti-HER2 mAb
H2Mab-119 (IgG1, kappa) by immunizing mice with HER2 ectodomain (HER2ec) produced in glioblastoma LN229 cells.38 This
antibody is judged as a pan-HER2 mAb that reacts with HER2
regardless of the cell type, because its FACS staining patternis indistinguishable from that of trastuzumab (Figures 1A and
1B). In flow cytometric analysis using saturating concentrationsof each antibody, H2Mab-119 and trastuzumab brightly stained
CHO-K1 cells expressing exogenously introduced HER2 (CHO/
HER2) (Figure 1A) as well as BT-474 and SK-BR-3 breast cancer
cell lines that are known to express endogenous HER2 (Figure 1B).39 They also recognized immortalized normal cell lines,
including HEK293T (embryonic kidney), HaCaT (keratinocyte),
and MCF 10A (mammary gland epithelial cell), but not triplenegative breast cancer (TNBC) cell line, MDA-MB-468 cells
known to be HER2-negative (Figure 1B).40
Next we aimed at obtaining anti-HER2 mAbs that recognize
HER2 only when expressed on cancer cells. We incorporated
differential screening protocol in the hybridoma selection
step34 and succeeded in establishing a few mAbs that recognized HER2-positive cancer cell lines, but not HER2-expressing
normal cells. As shown in Figure 1B, one of the clones, H2Mab214 (mouse IgG1, kappa), reacted with BT-474 and SK-BR-3
cells, but not with HEK293T, HaCaT, and MCF 10A cells. To eliminate the possibility that the differences in binding behavior
between H2Mab-214 and other two antibodies on flow cytometric analysis were due to differences in their affinity for HER2,ELISA-based affinity measurement was performed for these antibodies. The results showed that the KD values of trastuzumab,
H2Mab-119, and H2Mab-214 for recombinant soluble HER2ec
were 0.31 nM, 0.30 nM, and 0.53 nM, respectively (Figure 1C),
with the value of trastuzumab was comparable to that previously
determined by a similar method.41 As the KD value of H2Mab-214
was not significantly different from those of the other two antibodies, their binding behavior was shown to be affinity-independent. Binding of H2Mab-214 was also observed with CHO cells
overexpressing HER2 (Figure 1A), suggesting that the forced
high-level expression in this normal cell line may render HER2
a H2Mab-214 positive, cancer-like trait to some extent. Taken
together, these results suggest that H2Mab-214 exhibits specificity to HER2-positive breast cancer cells.
Structure 32, 536–549, May 2, 2024 537

llArticleA

H2Mab-119

400

200

300
200

100

100

0 0 1 2 3 4 5 6 0 0 1 2 3 4 5 6
10 10 10 10 10 10 10 10 10 10 10 10 10 10

H2Mab-214

CHO/HER2

300

400

200

300
200

100

Fluorescence intensity

100

0 0 1 2 3 4 5 6 0 0 1 2 3 4 5 6
10 10 10 10 10 10 10
10 10 10 10 10 10 10

CHO-K1

Overlay

CHO/HER2

300

400

200

300
200

100

100

0 0 1 2 3 4 5 6
10 10 10 10 10 10 10

0 0 1 2 3 4 5 6
10 10 10 10 10 10 10

CHO/HER2

400
300
200
100
0 0 1 2 3 4 5 6
10 10 10 10 10 10 10

Fluorescence intensity

Fluorescence intensity

Fluorescence intensity

500

Cell number

300

CHO-K1
Cell number

Cell number

500

CHO/HER2

Cell number

Trastuzumab
CHO-K1B

Trastuzumab
BT-474

MDA-MB-468

SK-BR-3

Cell number

500
300

400
300

200

200

200

0 0 1 2 345
6
10 10 10 10 10 10 10

0 0 1
2
3
4
5
6
10 10 10 10 10 10 10

400

HaCaT

MCF 10A
500

400

300

300

200

400
300
200

200

100

100

100

HEK293T
500

300

100

100

0 0 1 2 3 456
10 10 10 10 10 10 10

0 0 1 23
4
5
6
10 10 10 10 10 10 10100

0 0 1 2 3 456
10 10 10 10 10 10 10

0 0 1 23
4
5
6
10 10 10 10 10 10 10

Fluorescence intensity

H2Mab-119
BT-474

MDA-MB-468

Cell number

SK-BR-3
400

500
400

300

300

200

500

HEK293T

400

300

HaCaT

MCF 10A
500

300

300

200

400

400
300

200

200

200
100

100
0 0 1 23
4
5
6
10 10 10 10 10 10 10100

0 0 1 23
4
5
6
10 10 10 10 10 10 10

200
100

100

0 0 1 23
4
5
6
10 10 10 10 10 10 10100

0 0 1 20 0 1 2 3 4
3
4
5
6
5
6
10 10 10 10 10 10 10
10 10 10 10 10 10 10

0 0 1 23
4
5
6
10 10 10 10 10 10 10

Fluorescence intensity

H2Mab-214
BT-474

MDA-MB-468

Cell number

500
400

SK-BR-3

300

300

200

200

100

100

HEK293T

HaCaT

MCF 10A
500

400
300

400

300

300
200
100
0 0 1 23
4
5
6
10 10 10 10 10 10 10

0 0 1 23
4
5
6
10 10 10 10 10 10 10

200
100

0 0 1 23
4
5
6
10 10 10 10 10 10 10

300

200

200
100

100

0 0 1 20 0 1 2
3
4
5
6
3
4
5
6
10 10 10 10 10 10 10 10 10 10 10 10 10 10

0 0 1 2 3 4 5
610 10 10 10 10 10 10

Fluorescence intensity

C

Trastuzumab

KD = 0.31 (nM)2

R = 0.9706

H2Mab-119

KD = 0.30 (nM)2

R = 0.9877

H2Mab-214

KD = 0.53 (nM)R2 = 0.9940

Figure 1. Binding analysis of anti-HER2 antibodies(A) CHO-K1 and CHO/HER2 cells were treated with 10 mg/mL of trastuzumab, H2Mab-119, and H2Mab-214 or buffer control, and analyzed in flow cytometry.
(B) BT-474, MDA-MB-468, SK-BR-3, HEK293T, HaCaT, and MCF 10A cells were treated with 10 mg/mL of trastuzumab, H2Mab-119, H2Mab-214 or buffer control
and analyzed in flow cytometry. The black line represents the negative control (blocking buffer).
(C) ELISA-based affinity measurement. The binding affinity of trastuzumab, H2Mab-119, and H2Mab-214 for HER2ec was measured by ELISA. Serially diluted
antibody solutions (0.003–50 mg/mL) of each antibody were incubated with wells coated with the HER2ec, followed by incubation with peroxidase-conjugatedanti-human or anti-mouse antibodies. The optical densities were measured at 655 nm and plotted. KD values were calculated by fitting the binding isotherms
using the built-in one-site binding models in GraphPad PRISM 6. Data are from a single experiment performed in duplicate (mean ± SEM).

538 Structure 32, 536–549, May 2, 2024

ll
ArticleA

579-625 (trastuzumab)
intracellular region
kinase domain
IVextracellular region

II

I
23

III

218

342

511

652 675 720

987

1255

HER2-'N218
HER2-'N342
HER2-'N511
PA-tagB

CHO/
HER2-'N342

CHO/
HER2-'N218

C

CHO/
HER2-'N511

200

200

200

100

100

100

0.8

H2Mab
-214

Cell number

0 0
10

1

10

2

10

3

10

4

0 0
10

10

1

10

2

10

3

10

0 0
10

4

200

200

200

100

100

100

10

1

10

2

10

3

10

4

0.6
0.5
0.4
0.3
0.2

0

H2Mab
-119

10

0.7

OD 655 nm

NZ-1

10

0

10

1

10

2

10

3

10

4

0 0
10

10

1

10

2

10

3

10

0 0
10

4

200

200

200

100

100

100

0

10

0

10

1

10

2

10

3

10

4

0

10

0

10

1

10

2

10

3

10

4

0

10

1

10

2

10

3

10

4

0.1
0

10

0

1

10

10

2

10

3

10

4

Fluorescence intensity

OD 655 nm

1.0

E

1.8

0.9

1.6

0.8

1.4

0.7

1.2

0.6
0.5
0.4
0.3

OD 655 nm

D

1.0
0.8
0.6

0.2

0.4

0.1

0.2

0

0

Figure 2. Epitope mapping of H2Mab-214(A) Domain organization of HER2 and design of a series of N-terminal domain deletion mutants used for the flow cytometry analysis.
(B) Binding of H2Mab-214 and H2Mab-119 to the deletion mutants of HER2 transiently expressed on CHO-K1 cells. CHO-K1 cells expressing each mutant weretreated with NZ-1, H2Mab-214, H2Mab-119, or buffer control (black line), stained with Alexa Fluor 488-conjugated secondary antibodies, and analyzed in flow
cytometry.
(C–E) Evaluation of reactivity of H2Mab-214 toward various HER2-derived synthetic peptides by ELISA. 10 mg/mL of H2Mab-214 was incubated with wells coatedwith the synthetic peptides, followed by incubation with peroxidase-conjugated anti-mouse antibodies. The optical density was measured at 655 nm. Data arefrom one experiment (C) or from one triplicate experiment ((D) and (E); mean ± SEM). N.C., negative control.

Epitope mapping of H2Mab-214
Puzzled by the apparent cancer cell specificity of H2Mab-214 in
its HER2 recognition, we next explored its binding epitope. To
this end, we generated a series of N-terminal deletion mutantsof HER2 by successively removing domain I (DN218), domain II(DN342), and domain III (DN511) (Figure 2A). The comparable
expression levels of these mutants on CHO-K1 cells wereconfirmed by the staining with NZ-1 antibody against the

16-residue PA tag attached to the N-terminus of the truncation
mutants. H2Mab-214 exhibited full reactivity toward all mutants(Figure 2B), indicating that its epitope lies exclusively within
the domain IV. In contrast, the reactivity of H2Mab-119 wascompletely lost upon the deletion of the domain I alone, suggesting the critical involvement of domain I in its HER2 recognition.
To narrow down further the location of H2Mab-214 epitope, we
synthesized overlapping 20-mer peptides covering the domain
Structure 32, 536–549, May 2, 2024 539

llArticleA

Figure 3. Structural analysis of H2Mab-214

B

VL

I613

D618

R58

M611

Y30A(L1)

D56(H2)

D618
P617

P617
W100A(H3)
I613

P612
K615

F616
D54(H2)

W614

K615

VH

H2Mab-214

R95
(H3)

G31B(H1)
W614

C

A98(H3)

D

㻵

HER2
㻵㻵

domain I

C587:C596
C600:C623

㻵㻵㻵

P617
K615
P612 I613
D618
F616
W614
M611

VL
VH

㻵㼂

C626:C634
C630:C642

H2Mab-119
E

F
M611

I613

D618

K615

D618

M611
I613

F616
P612

(A) Close-up view of the antigen binding site in the
crystal structure of H2Mab-214 complexed with the
epitope peptide. H2Mab-214 is shown as a surface
model, and the epitope peptide is shown as a stickmodel with a transparent sphere model.
(B) Hydrogen bonding interactions observed
between H2Mab-214 and the epitope peptide.
Hydrogen bonds are denoted by dashed lines.
Water molecules are shown as sphere models.
(C) Crystal structure of H2Mab-119 complexed with
the HER2 domain I fragment. The domain I is shown
as a cyan cartoon model, and the H2Mab-119 isshown as a cartoon model with a transparent surface model. HER2 residues in contact with the
H2Mab-119 (within 4 Å) are indicated in magenta.
(D) Overall structure of the HER2 ectodomain (PDB
ID: 3n85). Met611-Asp618 region is colored in yellow. An expanded view of the region indicated by
the black box is shown on the right. The Ca atomsof the residues 611–618 are shown as spheres, anddisulfide bonds are shown as red stick models.
(E–H) Structural comparison of the Met611-Asp618
region in the HER2 ectodomain ((E); crystal structure and (G); schematic diagram) and the peptide
bound to H2Mab-214 ((F); crystal structure and (H);schematic diagram). See also Figure S2.

P617

and they do not contain sequence motifs
suspected to undergo chemical modifications including glycosylation, phosphoryGH
lation, and deamidation (Figure S1A).
Cancer cells
Normal cells
Also, this segment is partially overlappedwith the binding footprint of pan-HER2
reactive trastuzumab (residues 579–625)
M611-D618(Figure S1).9 Therefore, it is difficult toM611-D618
explain the cancer specificity of H2Mab214 simply by cancer-specific chemicalalterations or large domain-wise conformational change to expose its epitope.
Transmembrane
Transmembrane
To understand how H2Mab-214 distinguishes HER2 expressed on cancer cells
IV and tested their reactivity by ELISA. As shown in Figure 2C, from that on normal cells, we performed a crystallographic analH2Mab-214 showed strong reactivity with the peptide corre- ysis of H2Mab-214 in complex with its epitope peptide. To thissponding to residues 603–622. Subsequent analysis using the end, antigen-binding domain of H2Mab-214 was expressed as
second set of shorter peptides derived from this region revealed a small and hyper-crystallizable antibody fragment, Fv-clasp.42
that the linear segment Pro612-Asp618 of HER2 confers the H2Mab-214 Fv-clasp was crystallized in the presence of theminimally required binding epitope for H2Mab-214 (Figure 2D). excess amount of HER2 (611–618) peptide, and the complex
Finally, binding toward alanine-substituted HER2 peptides structure was solved at 1.75 Å resolution (Table S1; Figure S2A).
were evaluated to see the contribution of individual amino In the crystal structure, the Fv region has a lower averageacid. H2Mab-214 showed significantly reduced reactivity toward B-factor than the SARAH domain (average B-factors for the Fv
K615A and F616A peptides, indicating that Lys615 and Phe616 and SARAH domains are 47.2 and 69.6, respectively), and a clear
have major contribution to the recognition by H2Mab-214 electron density was observed throughout. At the antigen binding site, a clear electron density corresponding to the epitope(Figure 2E).
peptide was also observed, allowing us to build reliable models
for all eight residues of the peptide (Figure S2B). The bound
Crystallographic analysis of H2Mab-214 in complex withpeptide is in a compact U-shaped conformation (Figure 3A),
the epitope peptide
The epitope mapping of H2Mab-214 revealed that it recognized but unlike the typical turn conformation found in protein loops,
linear 7-residue peptide 612-PIWKFPD-618. This segment and the it is not maintained via intramolecular main chain hydrogenregions surrounding it are present in all major variants of HER2, bonds. Rather, the conformation is stabilized by intermolecular
P612

W614

P617

540 Structure 32, 536–549, May 2, 2024

W614

F616
K615

ll
ArticleTable 1. The main chain dihedral angles
Phi/Psi angles ( ) in the HER2
ectodomain structures(PDB: 6j71)

Phi/Psi angles ( ) in
the H2Mab-214 Fv-clasp/epitope peptide complex

residue(PDB: 3n85)

Pro61260.9/143.0

86.6/138.7

62.7/149.5

Ile613104.1/113.8

91.2/117.5

106.6/102.1

Trp614107.3/128.4

114.7/112.1

76.7/-38.3

Lys615138.3/166.5

133.8/170.7

109.5/-112.4

Phe616

141.6/152.4

147.6/157.1

110.6/161.9

Pro61778.1/160.6

66.4/159.1

53.0/125.4hydrogen bonding interactions formed between the residues
from the peptide and Gly31B (CDR-H1), Arg58, Arg95 (CDRH3), Ala98 (CDR-H3), and Trp100A (CDR-H3) of H2Mab-214 (Figure 3B). In addition, numerous inter- and intramolecular van der
Waals contacts formed throughout the peptide reinforce highly
complementary recognition interface that warrant high affinity
for such a short stretch of amino acids (Figure 3A). Among the
peptide residues, Lys615 and Phe616 appear to form particularly
important interactions: the side chain of Lys615 forms salt
bridges with two aspartic acid residues (Asp54 and Asp56) in
CDR-H2 (Figure 3B), while the side chain of Phe616 is inserted
into a deep pocket at the center of the antigen binding site andin contact with the surrounding residues (Figure 3A). These observations are in good agreement with the results of the alanine
scanning (Figure 2E).
We also conducted a crystallographic analysis for the panHER2 antibody H2Mab-119. As H2Mab-119 was found to retain
high binding affinity for the HER2 domain I fragment (Figure S2C),
the H2Mab-119 Fab was co-crystallized with the domain I fragment. In the complex structure solved at 1.69 Å resolution,
H2Mab-119 is in contact with two b-strands and multiple loopsof the domain I, indicating that H2Mab-119 recognizes threedimensional (i.e., non-linear) epitope, which is in contrast to
H2Mab-214 (Figures 3C and S2D).
Structural comparison of the epitope peptide bound to
H2Mab-214 and the corresponding region in publishedHER2 structures
HER2 domain IV has a stalk-like structure consisting of multiple
short b-sheets stabilized by ten disulfide bonds, and the H2Mab214 epitope sequence (Pro612 to Asp618) is located near its
C-terminus (Figure 3D). So far, ten HER2 ectodomain structurescontaining atomic models of the epitope region have been reported (i.e., PDB IDs: 1n8z, 3be1, 3n85, 6bgt, 6j71, 6oge, 7mn5,
7mn6, 7mn8, and 8ffj), of which 3n85 and 6j71 are relatively wellmodeled with no disordered regions in the domain IV (Figure S2E).
In all of these structures, the Pro612-Asp618 region assumes
an extended conformation, with its central segment (Ile613 toPhe616) assuming a b-strand sandwiched by two strands toform a b-sheet (Figure 3D, inset), although the loop regions preceding or following it (dashed ellipses in the inset in Figure S2E) havestructural diversity, some are unmodeled. This "canonical" conformation of the 612–618 segment (Figure 3E) is drastically different
from the U-shaped conformation seen in the same peptide boundto H2Mab-214 (Figure 3F). Analysis of the residue-wise main chain
dihedral angles for the H2Mab-214-captured peptide revealed

large divergence from the values obtained for the published ectodomain structures, especially at Trp614, Lys615, and Phe616(Table 1). Such fundamental difference in the conformation tells
us that the 612–618 segment needs to be dislodged or "pulledout" from the b-sheet to be recognized by H2Mab-214, suggestingthat the HER2 molecules reactive with H2Mab-214 must undergo
local unfolding near its epitope region (Figures 3G and 3H).
Effect of forced unfolding of HER2 by DTT treatment on
the reactivity of anti-HER2 mAbs
Structural studies suggested that H2Mab-214 preferentially recognizes HER2 domain IV when it is structurally compromised.
However, because our crystal structure is not in complex with
the HER2 ectodomain, but with the epitope peptide, we do notknow whether H2Mab-214 indeed recognizes misfolded HER2
on the cell surface. Therefore, to validate this hypothesis, wesought to artificially disrupt the tertiary structure of HER2 on
normal cells and investigated antibody bindings to these cells.
HER2 has many disulfide bonds in its extracellular region contributing to the overall structural stabilization at varying degree. To
break the disulfide bonds of HER2 present on HEK293T cells,
cells were cultured in the media containing 1 mM DTT for 1, 5,
and 24 h, and reactivities of the antibody were investigated byflow cytometry (Figure 4A).43 Remarkably, binding of H2Mab214 was increased more than 2-fold after the 1-h treatment
with DTT, indicating that the number of epitope-bearing species
increased due to the partial reduction of disulfide bonds. Furthermore, this increase was transient because the binding becameless pronounced after 5 h and returned to the basal level (without
DTT treatment) after 24 h. In sharp contrast, H2Mab-119 exhibited completely opposite behavior to the DTT-treatment; thebinding was reduced by >95% after 1-h DTT treatment, followedby the gradual recovery over time, returning to the basal level after
24 h (Figure 4B). These observations are consistent with the ideathat brief incubation with DTT induces partial disruption of the tertiary structure of HER2, which is favored by H2Mab-214 but disfavored by H2Mab-119, and that the normal structure is restored
as the effective concentration of DTT goes down by oxidation
during the cell culture at 37 C.
To generalize the aforementioned notion, reactivities of four
regular anti-HER2 mAbs, H2Mab-19 (mouse IgG2b, kappa,
epitope: domain III),44 H2Mab-181 (mouse IgG1, kappa, epitope:
domain III),45 H2Mab-41 (mouse IgG2b, kappa, epitope: domainIV),46 and trastuzumab (humanized IgG1, kappa, epitope:
domain IV), with HEK293T cells were investigated 1 and 24 hafter the addition of 1 mM DTT. As in the case of H2Mab-119,
all antibodies showed a markedly reduced reactivity with thecells at 1 h, and that was nearly recovered at 24 h (Figure S3).
The decreased binding of non-cancer-specific mAbs with epitopes in different domains upon addition of DTT suggests thatstructural disruption occurred throughout the HER2 molecule.
These results again highlight the unique character of H2Mab214 in that it recognizes structurally disrupted HER2.
If the reactivity of H2Mab-214 is governed by the partiallyunfolded nature of HER2 molecules on cell surface, the DTTinduced binding upregulation should be seen in any cell types.
In fact, DTT treatment of normal mammary gland epithelial cellline MCF 10A resulted in small but statistically significant(p < 0.05) increase in the reactivity of H2Mab-214 (Figure 4C).
Structure 32, 536–549, May 2, 2024 541

llArticleAB

H2Mab
Mab-119
119

0.005

0

10

2

10

3

10

4

10

0.009
0.006

Control051000

0h:DTT(-)
1h
5h
24h

0.012

H2Mab-214

MFI(median)

0.010

600
500
400
300
200
100
0

Unit Area

0h:DTT(-)
1h
5h
24h

MFI(median)

Unit Area

0.015

Mab-214
214
H2Mab

6

Fluorescence intensity

0.003

12

18

0

24

time (hr)800

H2Mab-119

600
400
200

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0
10

2

10

3

10

4

10

5

0

6

18

24

D
***

2.0
1.5

Fold change relative to control

2.5

MCF 10A

*

1.0
0.5
0.0
control DTT(-) DTT(+) DTT(-) DTT(+)
H2Mab-214

SK-BR-3

90

**

80
70

*

30
20
10
0

control DTT(-) DTT(+) DTT(-) DTT(+)
H2Mab-214

H2Mab-119

H2Mab-119

FE
1. C511:C520
2. C515:C528
3. C531:C540
4. C544:C560
5. C563:C576
6. C567:C584
7. C587:C596
8. C600:C623

Fold change relative to wild-type

Fold change relative to control

C

12time (hr)Fluorescence intensity

2.5
2.0
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1.0
0.5
0.0

H2Mab-214 epitope
9. C626:C634

H2Mab-119
Wild-type

Trastuzumab

H2Mab-214

C511A/C520A C567A/C584A C587A/C596A
C600A/C623A C626A/C634A C630A/C642A

10. C630:C642
Figure 4. Analysis of antibody binding upon disulfide bond breakage in HER2
(A and B) Flow cytometry analysis of DTT-treated HEK293T cells. HEK293T cells were pre-treated with 1 mM DTT for 1, 5, or 24 h and then stained with H2Mab214 (A), H2Mab-119 (B), or buffer control, followed by flow cytometry analysis. Line graphs of the median florescence intensity (MFI) values are provided on theright side of the histograms.
(C and D) Effect of DTT treatment of MCF 10A (C) and SK-BR-3 (D) cells on antibody binding. MCF 10A and SK-BR-3 cells were either untreated or pre-treatedwith 1 mM DTT for 1 h and then stained with H2Mab-214, H2Mab-119, or buffer control, followed by flow cytometry analysis. Results are reported as fold change in
MFI compared with the buffer control. Data are mean +SEM of four independent experiments. p values were calculated using unpaired one-tailed t test. *p < 0.05,**p < 0.01. ***p < 0.001.
(E) HER2 domain IV structure extracted from 3n85. The disulfide bonds are indicated by red sticks.
(F) Binding of antibodies to the disulfide bond removal HER2 mutants. CHO-K1 cells transiently expressing each mutant were stained with H2Mab-119, trastuzumab, or H2Mab-214 and analyzed in flow cytometry. Relative impact of each mutation on binding is expressed as the fold change in the % positive cells fromthe wild-type after normalizing the expression level of each mutant using the NZ-1-stained cells. Signals from cells transfected with empty vectors were used todefine antibody-bound cell populations (regions including <1% of cells transfected with empty vectors were used for the calculation of % positive cells). Data aremean ± SEM of three independent experiments. See also Figure S3.

Furthermore, the same treatment also upregulated H2Mab-214
reactivity toward SK-BR-3 cells which were already positive for
H2Mab-214 binding before the DTT treatment (Figure 4D), indicating that there is a pool of H2Mab-214-negative HER2 molecules expressed on breast cancer cells which can be converted
to H2Mab-214-positive ones upon DTT treatment.
542 Structure 32, 536–549, May 2, 2024

Effects of targeted disruption of domain IV disulfide
bond on the epitope exposure
Although the DTT treatment facilitated the conversion of HER2
domain IV to the alternative conformation reactive with H2Mab214, it severely reduced the binding of all other mAbs, indicatingthat the DTT-treated HER2 has globally disturbed structure thatll
Articledoes not accurately capture the character of H2Mab-214reactive species on cancer cells. Considering the fact thatmost anti-HER2 mAbs do not show cell-type specificity like
H2Mab-214, cancer-specific HER2 structural alteration may be
restricted to domain IV, particularly the region encompassing
residues 612–618. We therefore set out to investigate whether
targeted local disruption of this region via removing individual disulfide linkage can mimic cancer-specific presentation of the
H2Mab-214 epitope. As the H2Mab-214 epitope is locatedbetween the 8th and 9th disulfide bonds in the domain IV(Figure 4E), we chose to remove the 6th to 10th disulfide
bonds (C567:C584, C587:C596, C600:C623, C626:C634, andC630:C642), which are relatively close to the epitope. As a control, the first disulfide bond (C511:C520) which is 55 Å awayfrom the epitope was also removed. To remove individual disulfide bond, the pairing cysteine residues were simultaneously replaced with alanine in the context of full-length HER2. Upon thetransient expression in CHO-K1 cells, their reactivities against
the anti-HER2 antibodies were evaluated by flow cytometry.
As expected, H2Mab-119 reacted with all mutants at levels
nearly equivalent to the wild-type, confirming the structural
integrity at the domain I in these domain IV-mutants. Trastuzumab showed greatly reduced reactivity with the C567A/C584A
and C587A/C596A mutants and moderately reduced reactivity
with the C600A/C623A mutant, but binding to other mutants remained intact (Figure 4F). The epitope for trastuzumab iscomposed of three loops, Pro579-Gln583 (loop 1), Asp592Phe595 (loop 2), and Lys615-Pro625 (loop 3) (Figure S1B). Structurally, the C567:C584 and C587:C596 disulfide bonds arelocated behind the loop 1 and 2, respectively, and appear to support the conformation of these loops, and the C600:C623 is
located in a linker connecting the loop 2 and 3. Thus, the reduced
binding of trastuzumab in their removal correlates well with thestructural observations. When the same set of HER2 mutants
were evaluated for the binding by H2Mab-214, however, they
showed increased binding at varying degrees (from 30 to90%), except for the pair located away from the epitope (i.e.,
C511:C520) (Figure 4F). This result indicates that the exposureof H2Mab-214 epitope does not require global misfolding of
HER2 but can be facilitated by any local destabilization of thestructure near the Pro612-Asp618 segment, which serves as a
"weak ankle" of HER2 that readily changes its conformation
into an alternative one. In the mutant lacking the C630:C642,
where structurally the impact of the mutation appears to besmall, the relatively high binding of H2Mab-214 can be attributed
not only to the disruption of the epitope conformation but also to
the elongation of the conformation at the mutated site. Since themutated site is in proximity to the membrane, this elongation
might enlarge the space between the cell surface and the
epitope, potentially increasing the antibody accessibility.
ADCC and CDC by H2Mab-214-mG2a-f against BT-474
and MDA-MB-468 cells
The high selectivity of H2Mab-214 for cancer cells could be a major advantage in its pharmaceutical application. Therefore, we
evaluated the potential application of H2Mab-214 in cancer therapy. Since H2Mab-214 is a mouse mAb (mouse IgG1, kappa), we
chose H2Mab-119, a representative pan-HER2 mouse mAb(mouse IgG1, kappa), as a control. Murine IgG1 subclass anti-

bodies have reduced binding to FcgR receptors and are closer
to human IgG4-based therapeutics, while murine IgG2a are
effector competent and closer to human IgG1 antibodies.17
Furthermore, a core fucose deficiency on the Fc N-glycan has
been shown to enhance the Fc binding to the FcgRIIIa on theeffector cells and the ADCC activity.47 Therefore, we produced
class-switched (from IgG1 to IgG2a) and defucosylated mAbs
to evaluate the antitumor activity of H2Mab-214 and H2Mab119. The class-switched and defucosylated mAbs (H2Mab214-mG2a-f and H2Mab-119-mG2a-f) were produced by
combining their VH with CH of mouse IgG2a (Figure S4A). We
confirmed that H2Mab-214-mG2a-f and H2Mab-119-mG2a-f
recognized BT-474, but not MDA-MB-468 cells (Figure S4B).
As a defucosylated control mouse IgG2a mAb, we used
281-mG2a-f (anti-hamster podoplanin [PDPN] mAb), which neverrecognized BT-474 nor MDA-MB-468 cells (Figure S4B).
We first investigated whether H2Mab-214-mG2a-f and H2Mab119-mG2a-f could exert ADCC against BT-474 cells. Target cells(BT-474 and MDA-MB-468) were labeled with 10 mg/mL calcein
AM. ADCC were determined by the calcein release into the medium after co-culture with mAbs and effector splenocytes from
BALB/c nude mice. As shown in Figure 5A, H2Mab-214-mG2a-f
showed ADCC activity (48.0% cytotoxicity) against BT-474 cells
more potently than did the control mouse IgG2a (4.4% cytotoxicity; p < 0.01) and 281-mG2a-f (4.8% cytotoxicity; p < 0.01),
and the activity was comparable to that induced by H2Mab119-mG2a-f (43.6% cytotoxicity). We also investigated complement-dependent cytotoxicity (CDC) activities48,49 of H2Mab214-mG2a-f and H2Mab-119-mG2a-f against BT-474 cells. As
shown in Figure 5B, both antibodies exhibited similar levels of
CDC in BT-474 cells (H2Mab-214-mG2a-f: 76.5% cytotoxicity,
H2Mab-119-mG2a-f: 72.1% cytotoxicity), which were significantly higher than that induced by control mouse IgG2a (9.5%
cytotoxicity; p < 0.01) and 281-mG2a-f (17.3% cytotoxicity;p < 0.01). There were no differences between H2Mab-119mG2a-f or H2Mab-214-mG2a-f and control mouse IgG2a or
281-mG2a-f in ADCC (Figure 5C) and CDC (Figure 5D) against
MDA-MB-468 cells. These results demonstrated that H2Mab214-mG2a-f exhibited ADCC and CDC comparable to H2Mab119-mG2a-f against HER2-positive BT-474 cells, despite its
reduced reactivity in flow cytometry (Figure 1).
Antitumor effects of H2Mab-214-mG2a-f in BT-474
xenografts
We next investigated antitumor effects of the antibodies in
BT-474 xenograft mouse models. H2Mab-119-mG2a-f, H2Mab214-mG2a-f, control normal mouse IgG, or 281-mG2a-f were
injected intraperitoneally on days 7, 14, and 19, after the
inoculation of BT-474. The tumor volume was measured ondays 7, 12, 14, 19, 22, and 26 after the inoculation. Administration of H2Mab-119-mG2a-f and H2Mab-214-mG2a-f significantly
reduced tumor volume compared to that of control normalmouse IgG in the BT-474 xenograft models after days 14 and19, respectively (Figures 6A and S5). The reduction in tumor volume on day 26 in mice treated with H2Mab-119-mG2a-f and
H2Mab-214-mG2a-f was 41% and 35%, respectively, compared
to mice treated with the control normal mouse IgG. In addition,
as shown in Figure 6B, tumors from the H2Mab-119-mG2a-f
and H2Mab-214-mG2a-f-treated mice weighed significantly
Structure 32, 536–549, May 2, 2024 543

llArticle
B

ADCC

CDC

(BT-474 cells; HER2-positive)(BT-474 cells; HER2-positive)

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Figure 5. H2Mab-119-mG2a-f- and H2Mab214-mG2a-f-mediated ADCC and CDC against
BT-474 (HER2-positive) and MDA-MB-468(TNBC) cells
Target cells (BT-474 and MDA-MB-468) werelabeled with 10 mg/mL calcein AM. ADCC and CDC
were determined by the calcein release into themedium.
(A and C) ADCC induced by H2Mab-119-mG2a-f,
H2Mab-214-mG2a-f, control mouse IgG2a or
281-mG2a-f with effector splenocytes against
BT-474 (A) and MDA-MB-468 (C) cells.
(B and D) CDC induced by H2Mab-119-mG2a-f,
H2Mab-214-mG2a-f, control mouse IgG2a or
281-mG2a-f with complements against BT-474(B) and MDA-MB-468 (D) cells. Values are shown asmean ± SEM. Asterisks indicate statistical significance (**p < 0.01; Welch’s t test). ADCC, antibodydependent cellular cytotoxicity; CDC, complementdependent cytotoxicity.

CDC(MDA-MB-468 cells; TNBC)(MDA-MB-468 cells; TNBC)

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544 Structure 32, 536–549, May 2, 2024

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success of trastuzumab has demonstrated
the utility of antibody drugs in cancer thern.s.
n.s.
apy, and mAbs have become one of theleading modalities in this field. As of 2018
(i.e., before COVID-19), half of the top 10
best-selling antibody drugs were reported
to be against cancer.50 More recently,
attention has focused on the developmentof more innovative mAb-based therapeuticstrategies, such as antibody-drug conjugates (ADCs), chimeric antigen receptor(CAR)-T cell therapy, and bispecific antibodies. For HER2-positive metastatic
breast cancer, the ADCs, trastuzumab
less than those from control normal mouse IgG-treated mice emtansine and trastuzumab deruxtecan, have already been
(60% reduction; p < 0.01 and 72% reduction, respectively; developed. With the trend shifting toward such therapies withp < 0.01). The reduction in tumor volume and weight in H2Mab- high cell-killing activity, antibodies with high specificity for cancer
119-mG2a-f- and H2Mab-214-mG2a-f-treated mice was also cells are becoming increasingly important. In the development ofsignificant compared to 281-mG2a-f-treated mice (Figures 6A therapeutic antibodies against cancers, efforts are being made to
and 6B). As a control experiment, the antitumor effects were identify unique characteristics of cancer cells. A major strategy isalso investigated in the MDA-MB-468 xenograft models. As to identify molecules that are highly expressed on the surface ofa result, no difference was observed between H2Mab-119- certain types of cancer cells and have a high cancer cell/normal
mG2a-f or H2Mab-214-mG2a-f and control normal mouse IgG cell ratio as HER2. However, many of the molecules that areor 281-mG2a-f in both tumor volume (Figure S6A) and weight abnormally expressed on cancer cells have already been identi(Figure S6B), confirming that the antitumor effects observed in fied by extensive transcriptome and proteome analyses, making
the BT-474 xenograft models were HER2-dependent.
exploration for new cancer antigens very challenging. In addition,
Tumors that were resected from mice on day 26 are demon- in this strategy, the target molecules are common autoantigens
strated in Figures 6C and S6C. The body weight loss was not that are also expressed on normal cells, raising concerns about
observed in both H2Mab-119-mG2a-f and H2Mab-214-mG2a-f adverse effects caused by antibody drugs.
In this study, we succeeded in developing the cancer-specifictreated mice (Figures 6D and S6D). The mice on day 26 about
BT-474 xenograft and MDA-MB-468 xenograft were demon- mAb targeting HER2. In the cell lines examined here, the reactivityof H2Mab-214 against cancer cells was clearly observed, whereas
strated in Figures S6E and S6F, respectively.
that against normal cells was not detectable even by flow cytometry, a highly sensitive detection system (Figure 1). Since we evalDISCUSSION
uated only a limited number of cell types, further investigation is
Trastuzumab is a pioneer in molecular targeted therapy and has needed to determine whether H2Mab-214 exhibits selectivity
been the most effective treatment for HER2-positive breast can- against various types of HER2-positive cancer cells. Our structural
cer for more than 20 years since its FDA approval in 1998.25 The and cell-based binding analyses strongly suggested that thellArticleA
Tumor volume (mm3)250

Normal mouse IgG
H2Mab-119-mG2a-f
H2Mab-214-mG2a-f
281-mG2a-f

200
150
100

*
n.s.
n.s.
n.s.

50
0

0

** **
* **
* **
n.s. **

10** : H2Mab-119-mG2a-f vs normal mouse IgG** : H2Mab-119-mG2a-f vs 281-mG2a-f** : H2Mab-214-mG2a-f vs normal mouse IgG** : H2Mab-214-mG2a-f vs 281-mG2a-f

20

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B

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20

H2Mab-214-mG2a-f

0281-mG2a-f
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Ig

(A) BT-474 cells (5 3 106 cells) were subcutaneously injected into BALB/c nude mice. On day 7(arrow), H2Mab-119-mG2a-f (n = 8), H2Mab-214mG2a-f (n = 8), control normal mouse IgG (n = 8), or
281-mG2a-f (n = 8) were injected intraperitoneally
into mice (5 mg/kg). Additional antibodies were injected on days 14 and 19 (arrows). The tumor volume was measured on days 7, 12, 14, 19, 22, and26 after the inoculation of BT-474. Values are presented as the mean ± SEM. *p < 0.05, **p < 0.01
(ANOVA and Tukey’s multiple comparisons test).
(B) Tumor weight (day 26) was measured fromexcised BT-474 xenograft tumors. Values are presented as the mean ± SEM. **p < 0.01 (Welch’st test).
(C) Appearance of BT-474 xenograft tumors fromthe H2Mab-119-mG2a-f, H2Mab-214-mG2a-f, control normal mouse IgG and 281-mG2a-f-treatedmice on day 26 (scale bar, 1 cm).
(D) Mice weight in H2Mab-119-mG2a-f, H2Mab-214mG2a-f, control normal mouse IgG and 281-mG2a-ftreated groups. n.s., not significant. See also
Figures S5 and S6.

-f

H2Mab-119-mG2a-f

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Tumor weight (mg)80

Figure 6. Antitumor activity of H2Mab-119mG2a-f and H2Mab-214-mG2a-f against BT474 xenograft

G

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conformation as the corresponding region
in the EGFR.53 By contrast, H2Mab-214 is
a new type of cancer-specific mAb that
directly recognizes the unique structuralfeature of the target. In the current crystal
structure, the epitope peptide (Met611Asp618) assumes U-shaped conformationDnestled in the binding pocket of H2Mab30
Normal mouse IgG
214. This is not compatible with the canonH2Mab-119-mG2a-f
ical conformation observed in native HER2n.s.
reported so far. As the segment is part of aH
Mab-214-mG
-f
2
2a
20
rigid 3-strand b-sheet in the native struc281-mG2a-fture, the HER2 species reactive with
H2Mab-214 must have undergone local
10
misfolding at this region to allow the accessof the antibody. Therefore, H2Mab-214reactive HER2 on cancer cells and0
H2Mab-214-non-reactive HER2 on normal
0
10
20
30
cells are chemically identical molecules
Days after inocculation
having a subtle conformational difference
present in a restricted region near the
preferential binding of H2Mab-214 to cancer cells is due to the Pro612-Asp618 (Figures 3G and 3H). Although we cannot rule
recognition of a structural feature that only HER2 on cancer cells out the possibility that such misfolding transiently occurs in a minor
has, namely the misfolded region. Previously, cancer-specific an- population of HER2 molecules on normal cells, the cancer-specifictibodies that appear to recognize structural features of target mol- misfolding of the Pro612-Asp618 segment appears to give special
ecules, anti-EGFR antibody mAb806 and anti-a4b7 integrin anti- advantage of cancer selectivity to H2Mab-214, particularly whenbody MMG49, have been reported.51,52 Their cancer specificity combined with the high total number of the HER2 molecule onis, however, thought to be linked to the global conformation of their HER2-positive cancer cells.
Proteins are inherently prone to misfolding. Homeostasis oftarget molecules rather than to local structural features. More specifically, their epitopes are expected to be hidden on normal cells membrane and secretory proteins is normally maintained by
but exposed on cancer cells for some reason. For mAb806, it quality control system in the endoplasmic reticulum (ER), but
has also been demonstrated by crystallographic analysis that the cancer cells are constantly exposed to a variety of stressors
antigen peptide bound to the antibody has almost the same such as oxidative stress, hypoxia, and decreased energy supply,
Structure 32, 536–549, May 2, 2024 545

llArticlewhich are known to promote production and accumulation of
misfolded proteins in the ER (called ER stress).54 We speculatethat in such an abnormal condition of the ER in cancer cells, proteins with minor structural defects (such as a misformation of adisulfide bond that does not impair overall domain stability orfunction) may escape the quality control system and are transported normally, presenting a cancer-specific "mark" to the immune system. Since our study showed that the conformational
integrity of HER2 at residues 612 to 618 is susceptible to thelack of surrounding disulfide bonds, the local misfolding of
HER2 may have stemmed from the disregulated redox systemin the ER of cancer cells. In breast cancer cells, it has been reported that the expression of protein disulfide isomerases(PDI), PDIA1, PDIA3, PDIA4, and PDIA6, is elevated, suggesting
an abnormal state of the redox system in the ER.55 Disruption ofdisulfide bond formation in cancer cells has also been proposed
for EGFR.53
As far as we are aware, only H2Mab-214 shows cancer
specificity among the many anti-HER2 antibodies developed
to date, including trastuzumab that recognizes a region very
close to the H2Mab-214 epitope. Therefore, the H2Mab-214
epitope (Pro612-Asp618 region) may be very special in thatit undergoes spatially confined misfolding to expose highly
antigenic peptide in cancer cells. Surprisingly, when we immunized mice with a synthetic peptide containing the H2Mab-214
epitope sequence, all mAbs obtained were found to show cancer specificity (data not shown). This suggests that priorknowledge about the misfold-dependent cancer neoantigen
sequence may be leveraged to obtain useful cancer-specificmAbs. By using such strategy, it may be possible to establish
H2Mab-214-like antibodies that are more suitable for therapeutic applications, such as fully human mAbs and high-affinity mAbs. As evidenced by the performance of trastuzumab,
the lower stalk region of HER2 should be a promising target
in terms of the therapeutic efficacy as well. In fact, eventhough not all HER2 molecules on cancer cells are H2Mab214 reactive, H2Mab-214 showed anti-tumor activity comparable to the pan-HER2 reactive H2Mab-119 (Figures 4 and
6). Since this study used a human breast cancer xenograftmodel in nude mice which lack intact immunity, further studies
using syngeneic model of HER2-expressing murine breast
cancers are needed to evaluate the contribution of whole immune system.
HER2 has a long history as a successful cancer therapeutictarget and has been well studied, yet its unique and cancer-specific structural defect that can be targeted by mAb had not been
discovered until now. Our results indicate that some moleculesthat had not been considered as good cancer-specific therapeutic target due to the widespread expression in many tissues may
in fact become viable drug target if they are found to undergo
cancer-specific local misfolding. It is hoped that the expansionof potential targets will facilitate the development of mAb therapies for cancers for which effective treatments have not yet
been found.

d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY

B Lead contact
B Materials availability
B Data and code availabilityd EXPERIMENTAL

MODEL AND STUDY PARTICIPANT
DETAILS
B Cell lines
B Animalsd METHOD DETAILSB Antibodies
B Flow cytometry
B Enzyme-linked immunosorbent assay (ELISA)B ADCC
B CDC
B Antitumor activity of H2Mab-119-mG2a-f and H2Mab214-mG2a-f in xenografts of BT-474 and MDA-MB468 cells
B Crystallization
B Data collection, structure determination, and refinement
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.str.
2024.02.007.
ACKNOWLEDGMENTS
We would like to thank the staff of the beamline at SPring-8 for their help with
X-ray data collection. This work was supported in part by JSPS KAKENHI grant
numbers JP21H02416 to T.A. and JP22K07224 to Y.K. from Japan Society for the
Promotion of Science, by the Research Support Project for Life Science and Drug
Discovery (Basis for Supporting Innovative Drug Discovery and Life Science
Research [BINDS]) from Japan Agency for Medical Research and Development(AMED) under grant numbers JP22ama121011 to J.T. and JP22ama121008 to
Y.K., and also supported by AMED under grant numbers JP22am0401013 toY.K., 23bm1123027h0001 to Y.K., and JP22ck0106730 to Y.K.
AUTHOR CONTRIBUTIONS
T.A. designed and performed experiments, analyzed the data, and wrote themanuscript. E.M., H.S., T.O., T.T., and M.K.K. performed experiments andanalyzed the data. J.T. and Y.K. conceived the experimental design, analyzed
the data, and wrote the manuscript. All authors contributed to the preparationof the manuscript.
DECLARATION OF INTERESTS
M.K.K. and Y.K. submitted a patent application related to this work: H2Mab214. Patent application number WO2022114163. (2021).
Received: August 20, 2023
Revised: January 10, 2024
Accepted: February 13, 2024
Published: March 8, 2024
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llArticleSTAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

Antibodies
Alexa Fluor 488-conjugated anti-mouse IgG

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FITC-conjugated anti-human IgG

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H2Mab-119

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H2Mab-19

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H2Mab-41

Kato et al.46

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H2Mab-181

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Chemicals, peptides, and recombinant proteinsrProtein A Sepharose Fast Flow

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Cat#A39240

HEK293T

ATCC

CRL-3216

CHO-K1ATCC

CCL-61

BT-474ATCC

HTB-20

SK-BR-3

ATCC

HTB-30

MDA-MB-468

ATCC

HTB-132

MCF 10A

ATCC

CRL-10317

HaCaT

CLS Cell Lines Service GmbHCat#300493-ACADEMIC

Charles River Laboratories

Strain Code: 194

pcDNA3.1 vector

ThermoFisher scientific

Cat#V79020

pCAG-Neo vector

FUJIFILM Wako Pure Chemical Corporation

Cat#163-25601

pCAG-Ble vector

FUJIFILM Wako Pure Chemical Corporation

Cat#164-25631

GraphPad PRISM 6GraphPad Software

https://www.graphpad.com/

GraphPad PRISM 8GraphPad Software

https://www.graphpad.com/

Deposited data

Experimental models: Cell lines

Experimental models: Organisms/strains
Mouse: BALB/c Nude
Recombinant DNA

Software and algorithms(Continued on next page)e1 Structure 32, 536–549.e1–e5, May 2, 2024

ll
ArticleContinued
REAGENT or RESOURCESOURCEIDENTIFIER

FlowJo ver.10

BD Biosciences

https://www.bdbiosciences.com/
en-us/products/software/
flowjo-v10-software

XDS

Kabsch60

https://xds.mr.mpg.de/

PHASER

McCoy et al.61

http://www.ccp4.ac.uk

PHENIX

Adams et al.64

https://phenix-online.org/

Coot

Emsley et al.63

https://www2.mrc-lmb.cam.ac.uk/
personal/pemsley/coot/

MOLPROBITY

Chen et al.65

http://molprobity.biochem.duke.edu/

PyMOL

Schrödinger, LLC

https://pymol.org/2/

RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Takao
Arimori (arimori@protein.osaka-u.ac.jp).
Materials availability
H2Mab-214 is available from Tohoku University Graduate School of Medicine.
Data and code availability
Coordinates for the structural models of H2Mab-214 Fv-clasp/epitope peptide complex and H2Mab-119 Fab/HER2 domain I complex have been deposited to the Protein Data Bank under PDB accession numbers 8jyq and 8jyr, respectively. All deposited data ispublicly available as of the date of publication. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
Cell lines
Chinese hamster ovary (CHO)-K1, BT-474, SK-BR-3, MDA-MB-468, HEK293T, and MCF 10A cell lines were obtained from the American Type Culture Collection (ATCC). HaCaT cell line was obtained from Cell Lines Service GmbH (Eppelheim). CHO/HER2 weregenerated by transfecting pCAG/PA-HER2-RAP-MAP into CHO-K1 cells using Lipofectamine LTX (Thermo Fisher Scientific, Inc.).
A few days after transfection, PA tag-positive cells were sorted by a cell sorter (SH800; Sony Corp.) using NZ-1, which was originallydeveloped as an anti-human PDPN mAb.56
CHO-K1 and CHO/HER2 were cultured in RPMI-1640 medium (Nacalai Tesque, Inc.) or in Ham’s F-12 medium (FUJIFILM Wako
Pure Chemical Corporation), and BT-474, SK-BR-3, MDA-MB-468, HEK293T, and HaCaT were cultured in DMEM medium (Nacalai
Tesque, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc.), 100 units/ml of penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B (Nacalai Tesque, Inc.). MCF 10A was cultured in Mammary Epithelial
Cell Basal Medium BulletKit (CC-3150, Lonza) supplemented with 100 ng/ml cholera toxin (Sigma-Aldrich). All of the above celllines were cultured at 37 C in a humidified atmosphere with 5% CO2.
ExpiCHO-S, Expi293F, and Expi293F GnT1- cell lines were obtained from Thermo Fisher Scientific. ExpiCHO-S cells were cultured
in ExpiCHO expression medium (Thermo Fisher Scientific), and Expi293F and Expi293F GnT1- cells were cultured in Expi293 expression medium (Thermo Fisher Scientific). Those cell lines were cultured in a shaker at 37 C shaker in a humidified atmosphere with
8% CO2.
Animals
The animal experiment for generation of anti-HER2 mAbs was approved by the Animal Care and Use Committee of Tohoku
University (Permit number: 2019NiA-001). Animal experiments for ADCC and antitumor activity were approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (approval no. 2023-001 and 2023-018). BALB/cnude mice were obtained from Charles River Laboratories and maintained and monitored as described previously.57 The
loss of original body weight was determined to a point >25% and/or a maximum tumor size >3,000 mm3 as humane endpoints
for euthanasia.

Structure 32, 536–549.e1–e5, May 2, 2024 e2llArticleMETHOD DETAILS
Antibodies
Anti-HER2 mAb, H2Mab-119 (mouse IgG1, kappa), was established as previously described.38 H2Mab-214 (mouse IgG1, kappa) wasestablished by the same strategy. In brief, BALB/c mice were immunized with recombinant HER2-extracellular domain produced by
LN229 cells together with Imject Alum (Thermo Fisher Scientific, Inc.). After several additional immunizations, spleen cells were fusedwith P3U1 cells. The culture supernatants of hybridomas were screened using enzyme-linked immunosorbent assay with recombinant HER2-extracellular domain and flow cytometry. We finally established 278 clones of hybridoma and further performed thescreening of reactivity to HER2-positive breast cancers (BT-474 and SK-BR-3) and non-transformed normal epithelial cells including
HaCaT and MCF 10A using flow cytometry.
To identify VH/VL sequences, total RNA was prepared from the hybridoma cells using an RNeasy PLUS Mini Kit (Qiagen). The initial
cDNA strand was synthesized using the SuperScript IV Reverse Transcriptase (Thermo) via a priming oligo-dT, according to the manufacturer’s instructions. PCR amplification was performed with oligonucleotide mixtures of the degenerate primer and the constantregion primer for the heavy (g) and light (k) chains.58 PCR reactions were performed with HotStar Taq polymerase (Qiagen). The PCR
products were purified, subcloned into the pCR4-TOPO vector (Thermo), and subjected to nucleotide sequencing.
Variable (VH) and constant (CH) regions of heavy chain cDNAs of H2Mab-119 and H2Mab-214 were subcloned into the pCAG-Neo
vector (FUJIFILM Wako Pure Chemical Corporation). Variable (VL) and constant (CL) regions of light chain cDNAs of H2Mab-119 andH2Mab-214 were subcloned into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation). To produce recombinant
H2Mab-119 and H2Mab-214, the vectors of heavy and light chains were transfected into ExpiCHO-S cells using the ExpiCHO Expression System (Thermo Fisher Scientific Inc.). H2Mab-119 and H2Mab-214 were purified using Ab-Capcher (ProteNova Co., Ltd.).
Bound antibodies were eluted with an IgG elution buffer (Thermo Fisher Scientific Inc.), followed by immediate neutralization of
eluates, using 1M Tris-HCl (pH 8.0). Finally, eluates were concentrated, after which 10 mM sodium phosphate buffer, pH 7.5,
150 mM NaCl (PBS) was used to replace with the elution buffer using Amicon Ultra (Merck KGaA).
To generate H2Mab-119-mG2a-f and H2Mab-214-mG2a-f, we subcloned these VH cDNA and CH of mouse IgG2a into the pCAG-Ble
vector. These VL cDNA and CL cDNA of mouse kappa light chain were also subcloned into the pCAG-Neo vector. The vectors for
the H2Mab-119-mG2a or H2Mab-214-mG2a were transduced into BINDS-09 (FUT8-knockout ExpiCHO-S) cells, and recombinant
mAbs were produced using the ExpiCHO Expression System. H2Mab-119-mG2a-f and H2Mab-214-mG2a-f were purified using
Ab-Capcher. Preparation of 281-mG2a-f (defucosylated anti-hamster PDPN mAb, control defucosylated mouse IgG2a) was previously described.59 Trastuzumab (Herceptin) was obtained from Chugai Pharmaceutical Co., Ltd (Tokyo, Japan).
Flow cytometry
CHO-K1, CHO/HER2, BT-474, SK-BR-3, MDA-MB-468, HEK293T, HaCaT, and MCF 10A cells were obtained using 0.25% trypsin
and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells were treated with primary mAbs (H2Mab-119,
H2Mab-214, H2Mab-119-mG2a-f, H2Mab-214-mG2a-f, or trastuzumab) or blocking buffer (control; 0.1% BSA in PBS) for 30 min at4 C. Then, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000; Cell Signaling Technology, Inc.) or
FITC-conjugated anti-human IgG (1:1,000; Sigma-Aldrich for trastuzumab) for 30 min at 4 C.
To analyze the antibody binding to DTT-treated cells, HEK293T, SK-BR-3, and MCF 10A cells were cultured in the media containing 1 mM DTT for 1, 5, 24 hours. The cells were then washed repeatedly with PBS to remove DTT and treated with 0.5 mg/ml of trastuzumab or 10 mg/ml of H2Mab-119 (mouse IgG1, kappa), H2Mab-214 (mouse IgG1, kappa), H2Mab-19 (mouse IgG2b, kappa),
H2Mab-181 (mouse IgG1, kappa), or H2Mab-41 (mouse IgG2b, kappa) for 1 hour on ice, followed by treatment with 5 mg/ml of Alexa
Fluor 488-conjugated anti-human IgG or anti-mouse IgG for 45 min on ice.
For the assessment of antibody binding to HER2 mutants, CHO-K1 cells were transiently transfected with vectors encoding various
HER2 mutants or empty vector using X-tremeGENE HP DNA Transfection Reagent (Merck KGaA). The cells were stained with antibodies using the same procedure as for the DTT-treated cells.
All fluorescence data were collected using SA3800 Cell Analyzer (Sony Corp.) and analyzed using FlowJo (BD Biosciences).
Enzyme-linked immunosorbent assay (ELISA)For the epitope mapping, NUNC Maxisorp 96-well plates (Thermo Fisher Scientific, Inc.) were coated with synthetic peptides(10 mg/ml; PepScreen by Sigma-Aldrich) or HER2ec (10 mg/ml) for 30 min at 37 C and blocked with Superblock reagent (Thermo
Fisher Scientific, Inc.) or 1% bovine serum albumin (Nacalai Tesque, Inc.) in 0.05% Tween 20 containing PBS (1% BSA/PBST) for
30 min at 37 C. H2Mab-214 (10 mg/ml) was then added to the plate and incubated for 30 min at 37 C. To detect bound antibodies,
horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibody (Agilent Technologies) diluted to 1:2,000 in 1% BSA/PBST
was added and incubated for 30 min at 37 C. We used ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.) for the enzymatic reactions. The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc.).
To determine the binding affinity of trastuzumab, H2Mab-119, and H2Mab-214 for HER2, NUNC Maxisorp 96-well plates (Thermo
Fisher Scientific, Inc.) were coated with HER2ec (1 mg/ml) for 30 min at 37 C and blocked with 1% BSA/PBST for 1 hour at 37 C.
Serially diluted antibody solutions (0.003 to 50 mg/mL, which is approximately 0.04 to 650 nM) were then added to the plate and incubated for 1 hour at 37 C. Detection of bound antibodies was performed as above using HRP-conjugated goat anti-human IgG antibody (Sigma-Aldrich, for trastuzumab) or HRP-conjugated rabbit anti-mouse IgG antibody diluted to 1:3,000 in 1% BSA/PBST. Using
e3 Structure 32, 536–549.e1–e5, May 2, 2024

ll
Articlethe values of OD655 at 25 min after initiating the enzyme reaction, KD values were calculated by fitting the binding isotherms using thebuilt-in one-site binding models in GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA).
ADCC
Spleens were aseptically removed from six female BALB/c nude mice (five-week-old, Charles River Laboratories, Inc.) Single-cellsuspensions were obtained using a cell strainer (352360, BD Biosciences). Erythrocytes were removed with the treatment of icecold distilled water. The splenocytes were used as effector cells.
H2Mab-119-mG2a-f and H2Mab-214-mG2a-f-mediated ADCC was assayed as follows. Target cells (BT-474 and MDA-MB-468)
were labeled with 10 mg/ml Calcein AM (Thermo Fisher Scientific, Inc.). The target cells (2 3 104 cells) were plated in 96-well plates(3860-096, AGC TECHNO GLASS Co.,Ltd.) and mixed with effector cells (effector/target cells ratio, 50), 100 mg/ml of H2Mab-119mG2a-f, H2Mab-214-mG2a-f, 281-mG2a-f (control defucosylated mouse IgG2a) or control mouse IgG2a (Sigma-Aldrich). Following incubation for 4.5 h at 37 C, the Calcein release into the medium was analyzed using a microplate reader (Power Scan HT; BioTek
Instruments, Inc.,) with an excitation wavelength (485 nm) and an emission wavelength (538 nm).
Cytotoxicity (% lysis) was calculated as follows: % lysis = (E - S)/(M - S) x 100, where ‘‘E’’ is the fluorescence in cultures of both
effector and target cells, ‘‘S’’ is the spontaneous fluorescence of only target cells, and ‘‘M’’ is the maximum fluorescence following thetreatment with a lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM EDTA, and 0.5% Triton X-100).
CDC
The Calcein AM- labeled target cells (BT-474 and MDA-MB-468, 2 3 104 cells) were plated in 96-well plates (3860-096, AGC
TECHNO GLASS Co.,Ltd.) and mixed with rabbit complement (final dilution 1:10, Low-Tox-M Rabbit Complement; Cedarlane Laboratories) and 100 mg/ml of H2Mab-119-mG2a-f, H2Mab-214-mG2a-f, 281-mG2a-f or control mouse IgG2a. Following incubation for
4.5 h at 37 C, Calcein release into the medium was measured as indicated above.
Antitumor activity of H2Mab-119-mG2a-f and H2Mab-214-mG2a-f in xenografts of BT-474 and MDA-MB-468 cells
BT-474 and MDA-MB-468 (5 3 106 cells) resuspended in DMEM and mixed with BD Matrigel Matrix Growth Factor Reduced (BDBiosciences) were subcutaneously injected into the left flank of BALB/c nude mice (Charles River Laboratories, Inc). On day 7
post-inoculation, H2Mab-119-mG2a-f (n=8), H2Mab-214-mG2a-f (n=8), 281-mG2a-f (n=8) or control mouse IgG (FUJIFILM Wako
Pure Chemical Corporation) (n=8) in PBS were intraperitoneally injected (5 mg/kg). On days 14 and 19, additional antibody injections
were performed. The tumor volume was measured on days 7, 12, 14, 19, 22, and 26 after the inoculation of cells. Tumor volumes were
determined as previously described.57
Crystallization
To generate H2Mab-214 Fv-clasp, VH (residues 1-113) and VL (residues 1-108) regions of H2Mab-214 were appended to SARAH
domain derived from human Mst1.42 Hinge-less Fc (residues 234-447 of human IgG1) was subsequently appended to the VL-SARAH.
The DNA fragments were subcloned into the pcDNA3.1 vector (Thermo Fisher Scientific, Inc.). VH-SARAH and VL-SARAH-Fc wereco-expressed in Expi293F cells (Thermo Fisher Scientific, Inc.) and purified with rProtein A Sepharose Fast Flow (Cytiva). The Fc
portion was digested by treatment with a His-tagged IdeS protease for 2 hours at 37 C, followed by removal of His-IdeS by NiNTA Agarose (QIAGEN). The sample was further purified by anion-exchange chromatography on a MonoQ 5/50 GL column (Cytiva)
equilibrated with 20 mM Tris, pH 8.0, and the resultant sample was concentrated by ultrafiltration using Amicon Ultra (Merck Millipore)and mixed with synthetic epitope peptide (residues 611-618 of HER2, MPIWKFPD) to be the final concentration of 5 mg/ml H2Mab214 Fv-clasp and 0.6 mM peptide. To prepare the recombinant H2Mab-119 Fab, the VH to CH1 region of the H2Mab-119 heavy chain
was subcloned into the pCAG-Neo vector and co-expressed with the H2Mab-119 light chain in ExpiCHO-S cells. H2Mab-119 Fab
was purified with Capto L (Cytiva) followed by Protein G Sepharose 4 Fast Flow (Cytiva).
The HER2 domain I (residues 1-216) with a C-terminal His-tag was subcloned into pcDNA3.4 (Thermo Fisher Scientific, Inc.) and
expressed in Expi293F GnT1- cells (Thermo Fisher Scientific, Inc.), followed by purification with Ni-NTA Agarose. For the crystallization, H2Mab-119 Fab was mixed with HER2 domain I at 1.5-fold molar excess and subjected to size-exclusion chromatography (SEC)
on a Superdex 200 Increase 10/300 GL column equilibrated with 20 mM Tris, 150 mM NaCl, pH 8.0 (Figure S3C). The purified sample
was concentrated to 5 mg/ml by ultrafiltration using Amicon ultra (Merck Millipore).
Crystallization screening was carried out using The Classic Suite (QIAGEN), Wizard Classic 1 & 2 (Rigaku), JCSG-plus (MolecularDimensions), and ProPlex (Molecular Dimensions) crystallization reagents by using the sitting-drop vapor diffusion method at 20 C.
Data collection, structure determination, and refinement
Crystals of the H2Mab-214 Fv-clasp/epitope peptide complex obtained under the condition of 0.1 M CHES pH 9.5, 20 % w/v polyethylene glycol (PEG) 8000 (JCSG plus, tube 1-7) and the H2Mab-119 Fab/HER2 domain I complex obtained under the condition of
0.1M Sodium citrate pH5.5, 20% w/v PEG3000 (JCSG plus, tube 1-2) were cryo-protected by well solution containing 15% PEG200
and 30% PEG3000, respectively.
Diffraction data were collected at 100 K at beamline BL44XU of SPring-8 (Harima, Japan). The data were processed and scaled
using X-ray Detector Software.60 Initial phases were determined by molecular replacement with PHASER61 in the CCP4 package62
using the crystal structures deposited in Protein Data Bank (PDB: 7cea for H2Mab-214 VH, 7bsc for H2Mab-214 VL, 7cea for SARAH
Structure 32, 536–549.e1–e5, May 2, 2024 e4llArticledomain, 1f3d for H2Mab-119 VH-CH1, 3wkm for H2Mab-119 VL-CL, and 4hrl for HER2 domain I) as search models. The structuralmodels were modified with COOT,63 refined with PHENIX,64 and validated with MolProbity.65 Data collection statistics and refinement parameters are summarized in Table S1.
QUANTIFICATION AND STATISTICAL ANALYSIS
All data are expressed as mean ± standard error of the mean (SEM). Welch’s t test was used for the statistical analyses in ADCC,
CDC, and tumor weight. ANOVA with Tukey’s post hoc test was used for the statistical analyses in tumor volume and mouseweight. GraphPad Prism 8 (GraphPad Software, Inc.) was utilized for the calculations. P<0.05 was considered to indicate a statistically significant difference.

e5 Structure 32, 536–549.e1–e5, May 2, 2024