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ARTICLE OPEN
Bispecific antibody simultaneously targeting PD1 and HER2
inhibits tumor growth via direct tumor cell killing in
combination with PD1/PDL1 blockade and HER2 inhibition
Chang-ling Gu
1
, Hai-xia Zhu
1
, Lan Deng
1
, Xiao-qing Meng
1
, Kai Li
1
, Wei Xu
1
, Le Zhao
1
, Yue-qin Liu
1
, Zhen-ping Zhu
1
and
Hao-min Huang
1
Immune checkpoint blockade has shown significant clinical benefit in multiple cancer indications, but many patients are either
refractory or become resistant to the treatment over time. HER2/neu oncogene overexpressed in invasive breast cancer patients
associates with more aggressive diseases and poor prognosis. Anti-HER2 mAbs, such as trastuzumab, are currently the standard of
care for HER2-overexpressing cancers, but the response rates are below 30% and patients generally suffer relapse within a year. In
this study we developed a bispecific antibody (BsAb) simultaneously targeting both PD1 and HER2 in an attempt to combine HER2-
targeted therapy with immune checkpoint blockade for treating HER2-positive solid tumors. The BsAb was constructed by fusing
scFvs (anti-PD1) with the effector-functional Fc of an IgG (trastuzumab) via a flexible peptide linker. We showed that the BsAb
bound to human HER2 and PD1 with high affinities (EC
50
values were 0.2 and 0.14 nM, respectively), and exhibited potent
antitumor activities in vitro and in vivo. Furthermore, we demonstrated that the BsAb exhibited both HER2 and PD1 blockade
activities and was effective in killing HER2-positive tumor cells via antibody-dependent cellular cytotoxicity. In addition, the BsAb
could crosslink HER2-positive tumor cells with T cells to form PD1 immunological synapses that directed tumor cell killing without
the need of antigen presentation. Thus, the BsAb is a new promising approach for treating late-stage metastatic HER2-positive
cancers.
Keywords: HER2; Trastuzumab; PD1 blockade; bispecific antibody; antibody-dependent cellular cytotoxicity (ADCC); PD1
immunological synapse
Acta Pharmacologica Sinica (2021) 0:1–9; https://doi.org/10.1038/s41401-021-00683-8
INTRODUCTION
The HER2/neu oncogene encodes an epidermal growth factor
receptor (EGFR)-related receptor, HER2, an orphan receptor whose
ligand has not yet been identified. HER2 dimerizes with other
HER2 family members to activate downstream signaling pathways
and thus plays important roles in cell growth [1,2]. It has been
shown that HER2 is overexpressed in 20%–25% of invasive breast
cancer patients and its overexpression is associated with more
aggressive diseases and poor prognosis [3,4].
Trastuzumab (Herceptin), a recombinant humanized monoclonal
antibody (mAb) against HER2 developed by Roche/Genentech, has
become the standard of care for patients with HER2-positive breast
cancer over decades [5–7]. However, the objective response rates to
trastuzumab monotherapy range between 12% and 34% and the
majority of patients who initially responded to trastuzumab generally
develop resistance or relapse after a year [8]. To this end, alternative
anti-HER2 antibody-based therapeutics including antibody-drug
conjugates, such as T-DM1 and DS8201s, and bispecific antibodies
(BsAbs) are being actively developed and have shown significant
clinical benefits in several cancer indications [9–12].
Programmed death-1 (PD1) is an inhibitory receptor expressed
in T and B cells as well as myeloid-derived cells [13–16]. Binding of
PD1 to its ligand PDL1 (CD274, B7-H1) inhibits T-cell proliferation
and cytokine production and, thus, turns them into a state of
exhaustion [17,18]. Anti-PD1 mAbs capable of blocking PD1 and
PDL1 have shown promising antitumor activity in a broad
spectrum of cancer types in preclinical studies and clinical trials
[19–25]. Nevertheless, only a fraction of patents (approximately
20%) achieved durable clinical responses after anti-PD1 antibody
monotherapy, while most patents either had no responses at all or
exhibited transient responses [26–30].
BsAbs are antibodies comprising two distinct antigen-
targeting domains and can be used in place of combination of
two mAbs. BsAbs have been extensively studied over years for
therapeutic use, because they can provide therapeutic benefits
that mAbs cannot [31–35]. A BsAb can bridge its two target
antigens and bring them into close proximity [36]. A BsAb can
retarget the effector cells, e.g., T cells, against tumor cells by
simultaneously binding to cell surface antigens expressed from
both cells and thus significantly activate the antitumor activities
of effector cells [37–39]. A BsAb can also ligate two different
receptors on the same cell and change intracellular downstream
signaling [40]. Using antibody engineering technology to create
a BsAb targeting HER2 and PD1,weaimedtosynergizethe
Received: 19 December 2020 Accepted: 14 April 2021
1
Sunshine Guojian Pharmaceutical (Shanghai) Co. Ltd., 3SBio Inc. Company, Shanghai 201203, China
Correspondence: Zhen-ping Zhu (zhuzhenping@3sbio.com) or Hao-min Huang (huanghaomin@3s-guojian.com)
www.nature.com/aps
©The Author(s) 2021
1234567890();,:
antitumor activities rendered by HER2 blockade and PD1/PDL1
blockade.
We report the creation of a BsAb by combining an anti-HER2
antibody IgG and a scFv from an anti-PD1 antibody (609A). The
BsAb retained its binding specificity for its original targets and was
able to direct T cells to engage HER2-overexpressing tumor cells,
resulting in PD1 synapse formation. In addition to the ability to
block HER2 signaling and induce antibody-dependent cellular
cytotoxicity (ADCC), the BsAb also activated antitumor immunity
through PD1/PDL1 blockade in vivo. Importantly, the BsAb
exhibited superior tumor cell killing activity in the presence of
peripheral blood mononuclear cells (PBMCs) or activated T cells
relative to combination of the two parental mAbs. Thus, our anti-
HER2×anti-PD1 BsAb represents a new strategy to enhance
antibody therapeutic efficacy through tumor-targeted immune
checkpoint blockade.
MATERIALS AND METHODS
Cell culture
The cell lines used in this study were obtained from American
Type Culture Collection (ATCC; Manassas, VA, USA) unless
otherwise indicated. The cells were cultured at 37 °C with 5%
CO
2
. Cells for protein expression: HEK293E cells were cultured in
FreeStyle 293 medium (Life Technologies, Rockville, MD, USA).
Cells for bioassays: NCI-N87 or BT474 cells were cultured in RPMI-
1640 supplemented with 10% FBS. PD1/CHO-S cells were cultured
in CD Forti-CHO supplemented with 1% Glutamax and 0.4% anti-
clumping agent. PD-L1 aAPC/CHO-K1 cells (Promega, Cat#J1252,
Madison, WI, USA) were cultured in 90% Ham’s F12 (Gibco
Cat#11765-054) with 10% FBS, 200 μg/mL hygromycin B (Gibco,
Cat#10687-010, Shanghai, China) and 200 μg/mL G418 sulfate
solution (Promega, Cat#V8091). PD1 effector cells (Promega,
Cat#J1250) were cultured in 90% RPMI-1640, supplemented with
10% FBS, 200 μg/mL hygromycin B, 500 μg/mL G418 sulfate
solution, 1% sodium pyruvate (Promega, Cat#11360070), and 1%
MEM nonessential amino acids (Gibco, Cat#11140050). Cells for
ADCC: NK92-CD16 cells (ATCC, Cat#pta8837) were cultured in
αMEM medium without nucleosides and with 2 mM glutamine,
0.1 mM 2-mercaptoethanol, 0.2 mM inositol, 0.02 mM folic acid,
12.5% FBS, 12.5% Horse Serum (ATCC, Cat#302040), and 100 units/
mL human recombinant IL-2. Cells for animal study: MC38 cells
(Jennio Biotech, a mouse colon cancer cell line, Guangzhou, China)
were cultured in DMEM with 10% FBS; JIMT-1 (Cobioer,
Cat#CBP60378, Nanjing, China), a breast cancer cell line, were
cultured in DMEM medium supplemented with 10% FBS. PBMCs
(Cat#SLB-HP050A) and CD3
+
beads-isolated T cells (Cat#SLB-
CD3T-10AN) were purchased from Sailybio (Shanghai, China) and
used when applicable.
Protein expression and purification
Constructs expressing the anti-HER2×PD1 BsAb variants were
generated using the pTT5 vector (NRC Biotechnology Research
Institute, Montreal, QC, Canada). The expression vectors were
transiently transfected into HEK293E cells using 1 μg/mL 25 kDa
linear PEI (Polysciences, Inc.). One day after transfection, valproic
acid (Sigma) was added to the cell culture at a final concentration
of 3 mM. On day 2 after transfection, medium comprising 10%
GlutaMAX, 10% 400 g/L glucose, and 80% freestyle 293 medium
was added to the cell culture to 10% of the total volume.
Conditioned medium was collected 5–6 days after transient
transfection.
BsAbs in the culture medium were purified by MabSelect SuRe
(GE) affinity columns using an Akta Avant 25 fast protein liquid
chromatography purification system. After equilibrating the
column with buffer A (25 mM sodium phosphate, 150 mM sodium
chloride, pH 7.4), the culture media containing BsAbs was loaded
into the column, which was then eluted with buffer B (100 mM
sodium citrate, pH 3.5) to collect the desired proteins. The eluted
proteins were neutralized with 1 M Tris-HCl at pH 9.0.
Flow cytometric analysis of BT474 cells and PD1/CHO cells
To measure the binding affinity of the anti-HER2×PD1 BsAb for
HER2-overexpressing cells, BT474 cells (1 × 10
5
/well in 96-well
plate) were incubated with three-fold serial dilutions of the BsAb
ranging from 3.3 pM to 200 nM in 200 μL PBS at 4 °C for 1 h. Cells
were washed three times with PBS and then incubated with FITC-
conjugated goat anti-human IgG (Jackson, Cat.#109-095-003) at
4 °C for 1 h. The cells were washed and resuspended in 200 μL PBS
and were analyzed on FACS (BECKMAN, Cytoflex).
To measure the binding affinity for PD1-overexpressing cells,
PD1/CHO cells were used instead.
Bridging ELISA
PD1-ECD proteins (in-house, 200 ng/mL) were coated in 96-well
plates (Thermo Fisher, Cat#439454) at 4 °C overnight. The plates
were washed with PBST (PBS containing 0.05% Tween-20),
blocked for 1 h with PBS containing 2% BSA and incubated with
three-fold serial dilutions of antibodies for another hour at 37 °C.
The plates were then washed three times and incubated with
2 µg/mL His-tagged HER2-ECD for 1 h at 37 °C. After washing, an
HRP-conjugated anti-6×HisTag mAb (Invitrogen, Cat#MA1-21315-
HRP) was added and incubated for 1 h at 37 °C. The plates were
washed and the reaction was developed with TMB substrates. The
plates were then read on a SpectraMax 190 reader (Molecular
Devices) at 450 nm.
Proliferation inhibition assay
BT474 cells, a HER2-overexpressing human breast cancer cell line,
were seeded at 5000 cells/well in a 96-well culture plate (Costar,
Cat#3599) supplemented with RPMI-1640+10% FBS, and incu-
bated overnight at 37 °C with 5% CO
2
. The next day, the cells were
incubated with serially diluted anti-HER2×PD1 BsAb or trastuzu-
mab ranging from 8 pM to 150 nM in a final volume of 200 μL/well.
The plates were incubated at 37 °C for 6–7 days and viability was
quantitated with Cell counting Kit-8 (Dojindo, Cat#CK04). The
plates were then read on a SpectraMax 190 reader (Molecular
Devices) at 450 nm.
PD1/PDL1 blockade bioassay
The assay was carried out following the manufacturer’s instruction
(Promega, Cat#J1250). Briefly, PD-L1 aAPC/CHO-K1 cells were
seeded at 4 × 10
4
cells/well at 100 μL in white 96-well plates
followed by cultured overnight in a 37 °C incubator with 5% CO
2
.
The next day, the supernatant was discarded and the PD-L1 aAPC/
CHO-K1 cells were incubated with three-fold serial dilutions of the
BsAb ranging from 0.04 to 300 nM and PD-1 effector cells (5 × 10
4
/
well) in 80 μL assay buffer (99% RPMI-1640 supplemented with L-
glutamine +1% FBS) for 6 h. Bio-Glo™Reagent (80 μL) was added
to each well and the bottom was sealed with opaque film. The
plate was incubated at ambient temperature for 5–30 min and
then luminescence was measured using a SpectraMax i3x. The
expression of luciferases under the control of NFAT transcriptional
response elements was measured as a readout in response to
PD1/PDL1 blockade.
ADCC assay
BT474 cells were seeded in 96-well flat-bottom plates at a density
of 1 × 10
4
cells/well at 50 μL in RPMI-1640 supplemented with 5%
FBS. NK92a cells (4 × 10
4
/well, Cat#pta8837) were added to each
well in the presence of serially diluted anti-HER2×PD1 BsAbs and
control antibodies for a final reaction volume of 150 μL, and the
plates were incubated for 3 h at 37 °C and 5% CO
2
. Three hours
later, 100 μL supernatant was transferred to new plates. To
monitor cell lysis, 50 μL LDH substrate was added to each well
containing the supernatant and the plates were incubated at
An anti-HER2xPD1 BsAb exhibited superior antitumor activity
CL Gu et al.
2
Acta Pharmacologica Sinica (2021) 0:1– 9
room temperature for 15 min. The plates were then read at 490
nm on a SpectraMax 190 reader (Molecular Devices). The % lysis
was converted from OD values according to the following formula:
(OD
sample
−OD
T
−OD
nk
)/OD
lysis
× 100%. The EC
50
was calculated
using GraphPad Prism 7 software (GraphPad Software). In the case
of T cells, pre-activated T cells with anti-CD3 antibody were used
instead of tumor cells.
Tumor cell killing assay
PDL1-overexpressing N87 cells (N87-PDL1) were diluted to 1 × 10
5
cells/mL in RPMI-1640 supplemented with 1% FBS. N87-PDL1 cells
(100 μL/well) were seeded into the wells of 96-well plates and
incubated overnight at 37 °C and 5% CO
2
. The next day, 2, 10, and
50 nM of antibodies [the BsAb, trastuzumab, an anti-PD1 mAb
(609A) and trastuzumab+609A] and controls were added to each
well containing 5000 PBMCs for a reaction volume of 200 μL/well,
and the plates were incubated at 37 °C and 5% CO
2
for 4 days.
Cell-Titer Glo reagent (50 μL/well) was added to the plates
followed by incubation at room temperature for 5–10 min. The
plates were then read on an MD SpectraMax i3 for luminescence
at 500 ms.
In the case of activated T-cell killing assays, NCI-N87 cells were
seeded in 96-well plates at 1 × 10
4
cells/well. The next day, 2 and 9
nM of antibodies and controls were added to each well containing
5000 activated T cells for a reaction volume of 200 μL/well, and the
plates were incubated at 37 °C and 5% CO
2
for 7 days. The plates
were then read on an MD SpectraMax i3 for luminescence.
Immunofluorescence assay
PD1-overexpressing Jurkat T cells were stimulated to enhance PD1
expression. The activated T cells were labeled with 200 nM Alexa
Fluor 488 (488)-conjugated anti-HER2×PD1 BsAb (T1) or 488-
conjugated anti-PD1 mAb (609A) (T2) for 1 h at room temperature
respectively. N87 tumor cells were labeled with 100 nM Alexa
Fluor 546 (546)-conjugated anti-HER2 mAb (19H6-Hu) in the
presence (A1) or absence (A2) of trastuzumab for 1 h at room
temperature, respectively. The 488-labeled T cells were then co-
cultured with 546-labeled N87 cells with the following formats T1
+A2 or T2+A1 in PBS for 30 min at room temperature followed by
transfer into 6-well plates pre-coated with poly-lysine prior to
fixation with 4% paraformaldehyde. Images were visualized with
an LCAch N 40×/0.55 PhP objective (IX53, Olympus).
Xenografted and syngeneic tumor models
Animal care and in vivo experiments were approved by the
institutional IACUC of Sunshine Guojian Pharmaceutical (Shang-
hai) Co. Ltd. and performed under approved protocols (approval
code for NCI-N87 xenograft model: AS-2018-058; for
MC38 syngeneic model: AS-2019-006). NCI-N87 xenograft tumor
models were established with 6-week-old female Balb/c nude
mice (purchased from Charles River Laboratories) by subcuta-
neous injection of 8 × 10
6
NCI-N87 cells mixed with 50% Matrigel.
MC38 syngeneic tumor models were established in 18–20 g
female PD-1 humanized mice (B6-Pdcd1
em1(hPdcd1)Vst
/Vst, Vital star
Biotech) by subcutaneous injection of 1 × 10
6
MC38 cells. When
tumors reached a volume of approximately 150 mm
3
, the animals
were randomly divided into groups and i.p. injected twice a week.
Tumor volume was measured twice per week and calculated using
the formula V=LW
2
/2 (where V=volume, L=length, and W=
width).
In the case of the JIMT-1 xenograft model, JIMT-1 tumors were
established in M-NSG mice (NOD.Cg-Prkdc
scid
Il2rg
em1
/Smoc)
(Model Organism, Cat# NM-NSG-001) by subcutaneous injection
of 8 × 10
6
JIMT-1 cells mixed with 50% Matrigel. When tumors
reached a volume of approximately 200–500 mm
3
,3×10
6
PBMCs
in suspension were intraperitoneally inoculated, and then the
animals were randomly divided into groups and treated as
described above.
Statistical analysis
All numerical data, except for mouse xenograft data, are
presented as the mean ± standard deviation. Mouse xenograft
data are presented as the mean ± standard error. Statistical
analysis was performed with GraphPad Prism 7 software and Excel.
Pvalues were calculated using a two-way ANOVA multiple
comparison test. In all tests, differences with Pvalues < 0.05 (*)
were considered to be statistically significant.
RESULTS
Construction and production of the anti-HER2×anti-PD1 BsAb
The anti-HER2 antibody, trastuzumab, and the anti-PD1 antibody,
SSGJ-609A (609A), were utilized as the building blocks to construct
the anti-HER2×anti-PD1 BsAb via the IgG-scFv or scFv-IgG fusion
format [41–43]. In this format, the scFv of one antibody was fused
via a flexible peptide linker [(GGGGS)n,n=0–5] to the N- or C-
terminus of the heavy chain of the other antibody. Various
constructs were examined for their expression levels in transient
mammalian culture and their bioactivities in terms of binding to
both HER2 and PD1. When the scFv of trastuzumab was fused to
either the N- or C-terminus of the heavy chain of the IgG scaffold
of 609A, it showed a significantly reduced binding affinity for
BT474 cells (a HER2-overexpressing breast cancer cell line), and
was much less potent in inhibiting proliferation of the tumor cells,
compared to trastuzumab (data not shown). We next used
trastuzumab as the IgG scaffold and fused it with the scFv of
609A. Between the two IgG/scFv fusion orientations examined, the
BsAb constructed with the 609A scFv fused to the N-terminus of
trastuzumab showed ~5-fold lower binding affinity for BT474 cells
than did the BsAb with the 609A scFv fused to the C-terminus
(Supplementary Fig. S1). Thus, the BsAb with the two copies of
609A scFvs fused to the C-terminus of trastuzumab, namely anti-
HER2×PD1 BsAb, was selected for further characterization (Fig. 1a).
As demonstrated by SDS-PAGE, SEC-HPLC, and differential
scanning calorimetry, the anti-HER2×PD1 BsAb exhibited favor-
able chemophysical properties as a drug candidate (Fig. 1b–d).
The anti-HER2×PD1 BsAb simultaneously bound to HER2 and PD1
comparable to the parent monoclonal antibodies
The anti-HER2×PD1 BsAb dose-dependently bound to HER2 and
PD1 as shown by ELISA. The EC
50
(the antibody concentration
required for 50% of maximum binding) of the BsAb for HER2 was
0.2 nM. This was comparable to that of trastuzumab, which was
0.22 nM. Similarly, the EC
50
of the BsAb for human PD1 was 0.14
nM, which was comparable to the EC
50
of the parental anti-PD1
mAb, 609A (0.15 nM, Fig. 2a, b). The BsAb also bound efficiently to
the receptors on the cell surface as shown by FACS analysis. The
EC
50
of the BsAb binding to BT474 cells was 1.64 nM, comparable
to trastuzumab, which bound to the same cells with an EC
50
of
1.56 nM. The EC
50
of the BsAb binding to PD1-overexpressing CHO
cells was 1.78 nM, which was comparable to the EC
50
of the anti-
PD1 mAb, 609A (1.62 nM, Fig. 2c, d). In addition to binding to PD1-
overexpressing cell lines, we also tested the ability of the BsAb to
bind to primary T cells. As expected, the BsAb was indeed capable
of binding to activated primary T cells (Supplementary Fig. S2). To
confirm that the BsAb can simultaneously bind to its two targets, a
bridging ELISA was performed and the results showed that the
BsAb was capable of crosslinking HER2 and PD1 with an EC
50
of
0.24 nM, whereas the crosslinking activity was not achieved with
the parental mAbs, trastuzumab and 609A (Fig. 2e).
The anti-HER2×PD1 BsAb retained the full biological activities of
the respective parental antibodies in cell-based assays
In tumor cell growth assays, the BsAb effectively inhibited the
proliferation of HER2-overexpressing BT474 cells at an IC
50
(the
antibody concentration inhibiting 50% of cell proliferation) of 0.59
nM on par with that of trastuzumab whose IC
50
was 0.5 nM
An anti-HER2xPD1 BsAb exhibited superior antitumor activity
CL Gu et al.
3
Acta Pharmacologica Sinica (2021) 0:1 – 9
-4 -2 0 2 4
0
1
2
3
HER2 binding ELISA
log[c on ce ntra tion ](n M )
OD
450
anti-H ER 2 × PD 1 Bs A b
tras tu zu m a b
BT474 binding FACS
log[concentration](nM)
MFI
-4 -2 0 2
0
20000
40000
60000 anti-HER2×PD1 BsAb
trastuzu m ab
-4 -2 0 2 4
0.0
0.5
1.0
1.5
2.0
2.5
PD1 binding ELISA
log[concentration](nM)
OD
450
anti-HER2×PD1 BsAb
609A
log[concentration](nM)
MFI
-2 0 2 4
0
5000
10000
15000 anti-HER2×PD1 BsAb
609A
PD-1/CHO binding FACS
-4 -2 0 2
0.0
0.5
1.0
1.5
2.0
2.5
bridging ELISA
log[concentration](nM)
OD450
anti-HER2×PD1 BsAb
trastuzu m ab
609A
ab c
de
Fig. 2 The anti-HER2×PD1 BsAb simultaneously bound to PD-1 and HER2. a The binding affinity of the BsAb for HER2 was measured by ELISA.
Trastuzumab was used as the positive control. bThe binding affinity of the BsAb for PD1 was measured by ELISA and compared to that of the
parental anti-PD1 mAb, 609A. cThe ability of the BsAb to bind to BT474, a HER2-overexpressing cancer cell line, was measured by FACS and
compared to that of trastuzumab. dThe ability of the BsAb to bind to PD1-overexpressing CHO cells was measured by FACS and compared to that
of the parental anti-PD1 mAb, 609A. eA bridging ELISA was set up such that PD1 proteins were coated on the plates followed by the sequential
addition of the indicated antibodies and His-tagged HER2 proteins. Anti-6×HisTag monoclonal antibody-HRP was added to visualize the positive
binders. The results confirm that the BsAb is capable of simultaneously crosslinking its two targets, HER2 and PD1.
ab
cd
anti-PD1 scFv
Trastuzumab
250
36
28
17
10
55
72
95
130
12M3 4
Fig. 1 The structure and properties of the anti-HER2×PD1 BsAb. a Schematics of the anti-HER2×PD1 BsAb structure. bSDS-PAGE showing
nonreduced and reduced anti-HER2×PD1 BsAb. Lane 1: nonreduced BsAb; Lane 2: reduced BsAb; Lane 3: nonreduced trastuzumab; Lane 4:
reduced trastuzumab; M: Molecular weight markers. cSEC chromatogram showing that the BsAb purified by a single-step protein A affinity
column had over 95% monomeric species. dDifferential scanning calorimetry (DSC) of the anti-HER2×PD1 BsAb showing that the antibody
has a T
onset
(the temperature at onset of melting) of 52.5 °C and T
m
1/2/3 (melting temperatures) of 59.2 °C/68.4 °C/ 83.5 °C, respectively.
An anti-HER2xPD1 BsAb exhibited superior antitumor activity
CL Gu et al.
4
Acta Pharmacologica Sinica (2021) 0:1 – 9
(Fig. 3a). Similarly, the BsAb potently blocked PD1/PDL1 cell
signaling with an IC
50
of 3.28 nM, which was comparable to that of
609A (0.89 nM, Fig. 3b). Thus, the BsAb retained its ability to inhibit
HER2-overexpressing tumor cell growth via HER2 blockade and to
activate T cells via PD1 blockade.
The anti-HER2×PD1 BsAb exhibited ADCC toward HER2-
overexpressing tumor cells but not T cells
ADCC plays an important role in trastuzumab-mediated tumor cell
killing. Thus, we tested the BsAb for ADCC toward cancer cells and
T cells, as we would like to confirm whether the scFvs (anti-PD1)
fused to the effector-functional Fc of an IgG (trastuzumab) by
linkers still possessed ADCC activity. The BsAb exhibited strong
ADCC toward BT474 tumor cells, comparable to that of
trastuzumab (Fig. 4a). By contrast, no ADCC toward T cells could
be detected, while a control antibody targeting major histocom-
patibility complex (MHC) I of T cells exhibited potent ADCC toward
T cells [44] (Fig. 4b). This result is in line with the previous finding
where a BsAb with anti-PD1 scFvs fused to the N-terminus of
cetuximab showed no ADCC toward T cells [39]. This suggests that
proper spacing between the cell surface receptors and the Fc
region of an IgG molecule or a favorable protein configuration as a
whole, such as an intact IgG format, might be a critical
determinant for initiating ADCC.
The anti-HER2×PD1 BsAb exhibits synergistic killing of cancer cells
in a PDL1 expression-independent manner ex vivo
Given that the BsAb can crosslink PD1 on T cells and HER2 on
tumor cells, we hypothesized that the BsAb may enhance tumor
cell killing by bringing T cells into the proximity of tumor cells and
potentially inducing PD1 synapse formation as seen in our
previous study [39]. We stably transfected NCI-N87 cells, a HER2-
overexpressing gastric cancer cell line that does not express PDL1
when cultured in vitro (data not shown), with a PDL1 construct to
obtain a PDL1/HER2 double-overexpressing cancer cell line (N87-
PDL1) that could potentially respond to trastuzumab and anti-PD1
mAbs. Addition of various concentrations (2–50 nM) of trastuzu-
mab to N87-PDL1 cells in the presence of human PBMCs dose-
dependently reduced the number of viable cancer cells relative to
antibody-free controls (Fig. 5a). Combinations of trastuzumab and
the anti-PD1 mAb, 609A, showed a killing effect similar to that of
trastuzumab alone. Importantly, treating cancer cells with the
BsAb resulted in a superior reduction of viable cancer cells
compared to that of combination of trastuzumab with 609A (P<
0.0001; Fig. 5a).
Interestingly, 609A alone did not enhance PBMC-mediated N87-
PDL1 cell killing, even though PDL1 was overexpressed by the
tumor cells. To confirm that PDL1 on tumor cells was not
responsible for the lack of killing and that the enhanced killing
ab
-2 -1 0 1 2 3
0
10000
20000
30000
40000
PD1/PDL-1blockadebioassay
log[concentration](nM)
Lum(RLU)
IgG1
anti-HER2×PD1 BsAb
609A
log[concentration](nM)
OD
450
-3-2-10123
0.0
0.5
1.0
1.5 anti-HER 2×PD1 BsAb
trastuz um ab
B T 4 7 4 in h ib itio n
Fig. 3 The anti-HER2×PD1 BsAb inhibited the proliferation of HER2-overexpressing tumor cells and blocked the PD1/PDL1 interaction in
cell-based bioassays. a The BsAb inhibited the proliferation of HER2-overexpressing BT474 cancer cells in a dose-dependent manner similar
to trastuzumab. bThe ability of the BsAb to block PD1/PDL1 signaling was measured and compared to that of the parental anti-PD1 mAb,
609A, using a PD1/PDL1 blockade cell-based assay, in which the expression of luciferases were monitored under the control of nuclear factor
of activated T cells (NFAT ) response elements in response to blockade of PD1/PDL1 signaling (Promega Cat#J1250). A nonspecific IgG1 was
used as the negative control.
-3 -2 -1 0 1 2
-20
0
20
40
60
log [co nc en tratio n]( nM )
Lysis%
trastu zum ab
anti-HER2×PD1 BsAb
ADC C towards BT474 cells
-3 -2 -1 0 1 2
0
20
40
60
log[concentration](nM)
Lysis%
MHCIIgG1
trastu zum ab
anti-HER2×PD1 BsAb
ADCC towards T Cells
ab
Fig. 4 The anti-HER2×PD1 BsAb retained ADCC toward tumor cells but not T cells. a The BsAb exhibited a potency in lysing tumor cells
similar to that of trastuzumab in the ADCC assay. bThe BsAb and trastuzumab failed to mediate ADCC toward T cells, whereas an anti-MHC1
IgG1 antibody showed strong potency in lysing T cells in a dose-dependent manner [44].
An anti-HER2xPD1 BsAb exhibited superior antitumor activity
CL Gu et al.
5
Acta Pharmacologica Sinica (2021) 0:1 – 9
mediated by the BsAb was not simply due to PD1/PDL1 blockade-
induced T-cell activation, we examined the killing of parental N87
cells that did not express PDL1 with activated Jurkat T cells in the
presence of various antibodies. Consistent with the PBMC results,
the BsAb effectively killed more tumor cells than did either
trastuzumab alone, or the combination of trastuzumab with 609A.
Anti-PD1 mAb alone, again, failed to induce measurable tumor cell
killing in the absence of PDL1 on tumor cells (Fig. 5b). This finding
suggests that the crosslinking of T cells with cancer cells by the
BsAb, in contrast to the effects of the anti-PD1 mAb alone, might
be sufficient to allow T cells to recognize and initiate the killing of
tumors without the need for presentation of tumor-specific
antigens to T cells.
We next demonstrated by FACS that the physical association
between HER2-overexpressing tumor cells and T cells was indeed
established specifically by the BsAb (Fig. 5c and Supplementary
Fig. S3). In addition, immunofluorescence microscopy showed that
the BsAb bridged T cells with tumor cells and led to formation of
PD1 synapses [45,46]. It is possible that T cell activity could be
significantly further enhanced by PD1 synapses, since multiple
PD1 synapses could be simultaneously formed on a single T cell.
By contrast, no apparent tumor–T cell associations were observed
when the cells were incubated with a mixture of trastuzumab and
609A (Fig. 5d). Taken together, the above findings suggest that
the physical ligation of cancer cells with T cells by the BsAb might
enhance T cell-mediated antitumor effects in a way that cannot be
achieved by simply mixing two mAbs together.
The anti-HER2×PD1 BsAb exhibits potent antitumor activities in
both xenograft and syngeneic animal models
We next evaluated the antitumor effects of each of the two arms
of the BsAb in vivo. First, we measured the efficacy of the BsAb
for HER2 blockade in an N87 xenograft mouse model. The BsAb
suppressed 75% of N87 tumor growth at a dose of 20 mg/kg on
day 28 after treatment. The antitumor efficacy is comparable to
that of trastuzumab which inhibited 80% of tumor growth at an
equal molar dose of 15 mg/kg (Fig. 6aandSupplementary
Fig. S5d).
Second, we analyzed the antitumor activity of the BsAb for
PD1 blockade in an MC38 tumor cell line-derived human
PD1 transgenic mouse model. The BsAb effectively inhibited
~85% of tumor growth at a dose of 13 mg/kg on day 17 post
abc
d
PD1
HER2
Anti-HER2xPD1 BsAb Trastuzumab+609A BsAb+609A
Dual color cells: 23.31% Dual color cells: 1.26% Dual color cells: 5.00%
DIC PD1 HER2 Merge
Anti-HER2xPD1
BsAb
609A
+
Trastuzumab
50nM
10nM
2nM
600,000
800,000
1,000,000
lum(R LU )
anti-HER2×PD1BsAb+pbmc
trastuzuma b+ anti-PD1 m A b + pb m c
trastuzumab+pbmc
anti-PD1mAb +pbm c
N87-PDL1+pbmc
N87-PDL1
N87-PDL1 inhibition
**** ****
****
9nM
2nM
400,000
600,000
800,000
lum(RLU)
anti-HER2×PD1BsAb + Tcell
trastuzumab+anti-PD1 mAb + T cell
trastuzum ab + T cell
anti-PD1mAb + T cell
N87+ Tcell
N87
N87 inhibition
*
**
Fig. 5 The anti-HER2×PD1 BsAb exhibited synergistic killing effects on cancer cells in a PDL1 expression-independent manner ex vivo. a
Human PBMCs were mixed with PDL1-overexpressing N87 tumor cells (N87-PDL1) in the presence of 2, 10, and 50 nM indicated antibodies
(the N87-PDL1/PBMC pair). bActivated Jurkat T cells were mixed with parental N87 tumor cells in the presence of 2 and 9 nM indicated
antibodies (the N87/T cell pair). With respect to the combination treatment comprising trastuzumab and 609A, equal molar amounts of
the two mAbs (2, 10, and 50 nM each) were combined and added to the cells. The number of viable cells was measured as relative
luminescence units (RLUs). The different treatment groups were as follows: N87-PDL1 (N87), N87-PDL1 (N87) tumor cells only; N87-PDL1
+PBMCs (N87+T cells), N87-PDL1 cells mixed with PBMCs (N87 cells mixed with T cells) in the absence of antibodies; Anti-PD1 mAb+PBMCs
(Anti-PD1 mAb+T cells), N87-PDL1 cells mixed with PBMCs (N87 cells mixed with T cells), and the anti-PD1 mAb, 609A; Trastuzumab+PBMCs
(Trastuzumab+T cells), N87-PDL1 cells mixed with PBMCs (N87 cells mixed with T cells) and trastuzumab; Trastuzumab+anti-PD1 mAb
+PBMCs (Trastuzumab+anti-PD1 mAb+T cells), combination of trastuzumab and 609A added to N87-PDL1 cells in the presence of PBMCs
(N87 cells in the presence of T cells); and Anti-HER2×PD1 BsAb+PBMCs (Anti-HER2×PD1 BsAb+T cells), the BsAb added to N87-PDL1 in the
presence of PBMCs (N87 cells in the presence of T cells). *P< 0.05, **P< 0.01, and ****P< 0.0001 by two-way ANOVA. cPD1-overexpressing
Jurkat T cells prelabeled with an Alexa Fluor 647-conjugated anti-PD1 mAb (Sinobiological, Cat#MM18) were mixed with N87 cells prelabelled
with an Alexa Fluor 546-conjugated anti-HER2 mAb (19H6-Hu) in the presence of the BsAb (left), the mixture of trastuzumab and 609A
(center), the BsAb plus 609A (right). The group in red (pseudo-color) (upper right corner) shows cells that have dual emission signals (MFI >
1×10
4
for FITC-A and MFI > 3 × 10
4
for APC-A), meaning that the two types of cells were associated together. The group in green (pseudo-
color) (upper left corner) shows T cells labeled with the Alexa Fluor 647-conjugated anti-PD1 mAb (MFI > 3 × 10
4
for APC-A). The group in
purple (pseudo-color) (lower right corner) shows N87 cells labeled with the Alexa Fluor 546-conjugated anti-HER2 mAb (MFI > 1 × 10
4
for FITC-
A). dImmunofluorescence microscopy showing costaining of PD1 (green) on T cells and HER2 (red) on N87 tumor cells in samples treated with
the BsAb or a mixture of the anti-PD1 mAb (609A) plus trastuzumab. Note that one tumor cell was ligated with two T cells, while a single T cell
was in contact with two tumor cells in the presence of the BsAb. The combination of 609A and trastuzumab failed to induce PD1 synapse
formation with T cells. The white arrows denote T cells (DIC channel), for which PD1 staining (green channel) were barely seen owing to the
rearrangement of PD1 during synapse formation.
An anti-HER2xPD1 BsAb exhibited superior antitumor activity
CL Gu et al.
6
Acta Pharmacologica Sinica (2021) 0:1 – 9
treatment, on par with that of the anti-PD1 mAb, 609A, where
the parent mAb also inhibited 85% of tumor growth at an equal
molar dose of 10 mg/kg on the same day (Fig. 6band
Supplementary Fig. S5e). Thus, these results indicated that the
anti-HER2×PD1 BsAb retained the antitumor efficacy of each of
its arms to a level comparable to that of their parental mAbs
in vivo. It is worth noting that no apparent toxicity associated
with the BsAb was observed, as the animal body weights kept
climbing over the entire course of the treatment (Supplemen-
tary Fig. S5a, b).
Since the BsAb does not cross-react with murine PD1 and HER2,
we chose a model system in which we introduced human PBMCs
into immune-compromised NOD scid gamma mice in order to test
the synergistic antitumor activity of the BsAb. Human PBMCs were
injected intraperitoneally following the establishment of JIMT-1
tumors in the mice. Although injection of human PBMCs into mice
bearing JIMT-1 tumors slightly reduced the body weights initially,
the body weights of all groups returned to baseline following
treatment (Supplementary Fig. S5c). We then tested the antitumor
efficacy of the BsAb in comparison with that of the combination of
the two parental mAbs. The anti-PD1 mAb, 609A, failed to show
antitumor effects on JIMT-1 tumors relative to the control, whereas
609A in combination with trastuzumab significantly suppressed
tumor growth compared to the monotherapy. Importantly, the
BsAb also potently inhibited JIMT-1 tumor growth comparable to
that of the combination of the two parent mAbs (Fig. 6cand
Supplementary Fig. S5f), indicating that the BsAb indeed exhibited
dual targeting functions in vivo.
DISCUSSION
The goal of our study was to combine traditional HER2-targeted
therapy with PD1/PDL1 blockade with a novel anti-HER2×PD1
BsAb to treat HER2-overexpressing solid tumors. The BsAb
comprises a full trastuzumab IgG with two copies of anti-PD1
(609A) scFvs symmetrically fused to the C-terminus of the heavy
chains. The orientation of the IgG and the scFvs was selected
based on the finding that the BsAb with scFvs fused to the N-
terminus exhibited five-fold reduced binding affinity for HER2
receptors on tumor cells relative to that of the BsAb with scFvs
fused to the C-terminus. This is in contrast to an anti-EGFR×PD1
BsAb we previously reported, where fusion of 609A scFvs to the N-
terminus of the cetuximab heavy chains was required to keep the
binding affinity of the BsAb for target cells intact, in comparison
with the parental mAb, cetuximab [39]. Our results emphasize that
the geometrical steric hindrance of a receptor on the surface of
tumor cells should be carefully assessed during the design of
tumor-associated antigen (TAA)-targeting BsAbs.
ADCC plays an important role in trastuzumab-mediated tumor
cell killing [47–49]. Ideally, the BsAb is expected to retain full ADCC
toward HER2-overexpressing tumor cells while sparing T cells from
the attack. Indeed, the BsAb comprising the intact trastuzumab
Fig. 6 The anti-HER2×PD1 BsAb exhibited potent antitumor effects in vivo. a A control (black circle), the BsAb (blue square and pink
triangle), and trastuzumab (green triangle) were i.p. injected into mice bearing NCI-N87 tumors at the indicated doses. Tumor volumes (mm
3
)
were measured at the indicated time points. bA control (black circle), the BsAb (green triangle and diamond), and the anti-PD1 mAb, 609A
(red square and triangle) were i.p. injected into human PD1 transgenic mice bearing mouse MC38 tumors at the indicated doses. cAn isotype
control (blue square), 609A (orange triangle), 609A plus trastuzumab (green diamond, 20 mg/kg each), and the BsAb (red circle) were injected
into M-NSG mice bearing JIMT-1 tumors in the presence of human PBMCs at the indicated doses. WT mice (black dot) were used as control.
Tumor volumes (mm
3
) were measured at the indicated time points. Mean ± SEM. *P< 0.05 by two-way ANOVA (the BsAb/PBMC vs Vehicle/
PBMC) and ****P< 0.0001 for the comparison of all indicated groups with the control group.
An anti-HER2xPD1 BsAb exhibited superior antitumor activity
CL Gu et al.
7
Acta Pharmacologica Sinica (2021) 0:1 – 9
IgG exhibited ADCC toward HER2-overexpressing tumor cells as
effectively as parental trastuzumab, whereas the 609A scFv moiety
failed to induce measurable ADCC toward T cells, even though the
BsAb could effectively bind to PD1 on T cells. Interestingly, we
recently reported that the anti-EGFR×PD1 BsAb with 609A scFvs
fused to the N-terminus of the cetuximab heavy chains also
showed ADCC specific to tumor cells but not to T cells, suggesting
that an intact tertiary configuration of an IgG might be a critical
determinant for induction of ADCC to kill target cells.
Surprisingly, the anti-PD1 mAb, 609A, in the presence of PBMCs
did not enhance the killing of PDL1-overexpressing tumor cells
(N87-PDL1) compared to that of PBMCs alone. This was further
supported by the finding that the combination of trastuzumab
with 609A did not increase the killing of N87-PDL1 cells relative to
that of trastuzumab alone in the presence of PBMCs, suggesting
that blockade of PD1/PDL1 signaling did not play an important
role in T cell-mediated tumor cell killing in this experimental
setting. To confirm this hypothesis, we performed tumor cell
killing assays with NCI-N87 cells, which do not express PDL1, in
the presence of activated T cells. Indeed, the addition of 609A did
not enhance killing compared to the addition of T cells alone, nor
did it enhance trastuzumab-mediated killing when administered
in combination with trastuzumab. In contrast, the BsAb exhibited
superior killing of NCI-N87 tumor cells relative to all other
treatments. These data imply that BsAb-mediated tumor cell
killing may not require presentation of tumor cell antigens to
T cells mediated by the MHC as seen in the case of CD3-targeting
bispecific T-cell engagers [50]. However, T cells activated by PD1
blockade may still rely on, at least to a certain degree, MHC-
dependent antigen presentation for target cell recognition and
killing. It is plausible that in addition to the TCR/CD3 complex,
crosslinking a TAA with a functional regulatory receptor of T cells,
such as PD1, might be sufficient to initiate T cell-dependent
target cell killing without the need for MHC-mediated antigen
presentation.
In addition to direct tumor cell killing, the BsAb-induced
engagement of tumor cells with T cells via HER2 facilitated the
formation of PD1 immunological synapses as seen for the anti-
EGFR×PD1 BsAb [39]. These results suggest that T-cell activation
by PD1 synapse formation and MHC-independent target cell
recognition by T cells might be common advantages of a BsAb
targeting PD1 and a TAA over the combination of two mAbs for
therapeutic use.
Recently, growing evidence has demonstrated that HER2 signaling
might be coupled with PD1/PDL1 signaling in HER2-overexpressing
tumors [51]. Suh et al. demonstrated that PDL1 expression was more
frequent in patients with positive HER2 expression after analysis of
over 250 resected tumor tissues. In support of this clinical finding,
they also showed that cell lines overexpressing HER2 express a higher
level of PDL1 [52]. Interestingly, Fan’s lab showed that targeting HER2
with trastuzumab upregulated the expression of PDL1 via upregula-
tion of IFNγproduction, which presumably suppressed antitumor
immune responses and led to drug resistance [53]. Thus, a BsAb
simultaneously targeting HER2 and PD1/PDL1 signaling pathways
might be more effective in treating HER2-overexpressing tumors than
the corresponding mAbs. Indeed, our BsAb and a mouse specific
BsAb, BsPDL1 × rErbB2, which targets PDL1 and HER2 [54], showed
enhanced antitumor activities relative to that of monotherapies with
mAbs at equal molar doses in vivo, although the latter is a
monovalent BsAb that did not inhibit tumor cell growth in vitro,
suggesting that combining HER2 and PD1/PDL1 blockades might
improve the treatment of HER2-overexpressing cancers.
In summary, we produced a new anti-HER2×PD1 BsAb. The
BsAb was much more cytotoxic to HER2-overexpressing tumor
cells in the presence of PBMCs or activated T cells than was
trastuzumab either alone or in combination with the parental anti-
PD1 antibody, independent of PDL1 expression. We postulate that,
in addition to direct HER2 inhibition and ADCC, as is the case with
trastuzumab, there are several novel mechanisms that may further
contribute to the enhanced cytotoxicity of the BsAb: (1) the
BsAb redirects T cells to the proximity of tumor cells and induces
direct tumor cell killing most likely in an antigen-presentation-
independent manner; (2) the BsAb can potentially activate T cells
in the tumor microenvironment via PD1/PDL1 blockade, and (3)
the BsAb is capable of further enhancing T cell cytotoxic activity
by the formation of PD1 immunological synapses. Thus, we
provide a new promising option for treating late-stage or
refractory tumors that overexpress HER2.
ACKNOWLEDGEMENTS
We thank Ya Zhang and Xia Dong for their excellent technical assistance. We would
also like to acknowledge expert services provided by the Protein Chemistry and
Analytical Department at Sunshine Guojian Pharmaceutical (Shanghai) Co. Ltd.
AUTHOR CONTRIBUTIONS
CLG and HXZ made the constructs, designed and performed in vitro experiments
including cell-based assays and analyzed data; LD and XQM designed and
performed microscopy including acquisition of time-lapse movies; KL performed
ADCC assays; WX purified proteins; LZ and YQL performed in vivo studies; ZPZ
reviewed and edited the manuscript; HMH supervised the study and wrote and
revised the manuscript.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41401-021-00683-8.
Competing interests: The authors declare no potential conflicts of interest. All
authors are employees of Sunshine Guojian Pharmaceutical (Shanghai) when the
study was carried out.
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© The Author(s) 2021
An anti-HER2xPD1 BsAb exhibited superior antitumor activity
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