Author Manuscript Published OnlineFirst on February 8, 2019; DOI: 10.1158/1078-0432.CCR-17-3123 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
A T-cell-engaging B7-H4/CD3 bispecific Fab-scFv antibody targets human breast
cancer
Akira Iizuka1, Chizu Nonomura1, Tadashi Ashizawa1, Ryota Kondou1, Keiichi
Ohshima2, Takashi Sugino4, Koichi Mitsuya5, Nakamasa Hayashi5, Yoko Nakasu5,
Kouji Maruyama3, Ken Yamaguchi6, Yasuto Akiyama1, 5*
1Immunotherapy Division, 2Medical Genetics Division, 3Experimental Animal Facility,
Shizuoka Cancer Center Research Institute, 4Division of Pathology, 5Division of
Neurosurgery, 6Office of the President, Shizuoka Cancer Center Hospital, 1007
Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan
Running title: B7-H4/CD3-bispecific antibody targeting breast cancer
Keywords: B7-H4, breast cancer, bispecific antibody, MHC-dKO NOG mouse
Address correspondence to: Yasuto Akiyama, M.D., Immunotherapy Division,
Shizuoka Cancer Center Research Institute, 1007 Shimonagakubo, Nagaizumi-cho,
Sunto-gun, Shizuoka 411-8777, Japan. Tel.: (+81) 559895222 (Ext. 5330), Fax: (+81)
559896085, e-mail; [email protected]
Conflict of interest
The authors declare that they have no conflicts of interest.
Word count:
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Translational relevance: 138
Abstract: 247
Total word count: 7778
Total number of figures and tables: 6
Quantity of supplementary materials: 9
Translational relevance
Despite the clinical success of immune checkpoint blockade antibodies against
advanced cancers, the response rate is approximately 20-40% even for PD-L1-positive
cancers and most cancer patients are unlikely to benefit from treatment. Other
therapeutic antibodies have been developed and evaluated in clinical trials in
combination with anti-PD-1 antibodies.
In this study, we manufactured anti-B7-H4/CD3 bispecific antibodies (BsAbs) based
on the Fab and scFv structure. We found that the B7-H4/CD3 BsAb had potent
cytotoxic activity against B7-H4-positive breast cancer cell lines in vitro, and in vivo
using a MHC-dKO NOG mouse model. Because B7-H4 is highly expressed
independently of HER2 or PD-L1 expression in breast cancers obtained by the
High-tech Omics-based Patient Evaluation for Cancer Therapy (HOPE) project, the
B7-H4/CD3 bispecific antibody may be a good therapeutic tool for immune checkpoint
blockade or anti-HER2 antibody-unresponsive cancer patients.
ABSTRACT
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Purpose: The B7 homolog 4 (B7-H4, VTCN1) is an immune checkpoint molecule
that negatively regulates immune responses and is known to be overexpressed in many
human cancers. Previously, we generated a mouse anti-human B7-H4 monoclonal
antibody that did not have a significant antitumor effect in vivo probably because of
molecule instability. In this study, we designed a B7-H4/CD3 bispecific antibody
(BsAb) and investigated its antitumor activity in vitro and in vivo using a humanized
mouse model.
Experimental Design: Complementary DNAs of the antibody-binding fragment
(Fab)-single-chain variable fragment (scFv) and scFv-scFv of the anti-B7-H4/CD3
BsAb were synthesized, and the BsAb antibodies were produced in HEK293 cells. The
anti-tumor activity against human breast cancer cells by human peripheral blood
mononuclear cells (hPBMC) with BsAb was measured by lactate dehydrogenase (LDH)
release in vitro, and in vivo using hPBMC transplanted major histocompatibility (MHC)
class I and class II-deficient NOG mice.
Results: hPBMCs with anti-B7-H4/CD3 BsAbs successfully lysed the human breast
cancer cell line MDA-MB-468 (EC50: 0.2 ng/ml) and other B7-H4-positive cell lines in
vitro. When BsAb was injected in a humanized mouse model, there was an immediate
and strong antitumor activity against MDA-MB-468, HCC-1954 and HCC-1569 tumors
and CD8+ and granzyme B+ CTL infiltration into the tumor, and there were no adverse
effects after long-term observation. CD8+ T cell depletion by an anti-CD8 antibody
mostly reduced the antitumor effect of BsAb in vivo.
Conclusions: An anti-B7-H4/CD3 bispecific antibody may be a good therapeutic
tool for patients with B7-H4-positive breast cancers.
INTRODUCTION
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Because of the recent success of immune checkpoint blocking antibodies, clinical
trials are underway to evaluate their efficacy in various cancers (1-4). However, a
majority of cancer patients are unlikely to benefit from anti- programmed death-1
(PD-1)/ PD-ligand 1 (PD-L1) antibody treatment because the response rate is
approximately 20-40% even in PD-L1-positive cancers.
In addition to these immunomodulatory receptor blockade therapies, other
modulating technologies have been developed (5,6). Major histocompatibility complex
(MHC) and T cell receptor (TCR) bypassed T cell cytotoxicity was first reported in
1985 (7), and over the past 3 decades, CD3 bispecific antibodies (BsAbs) and chimeric
antigen receptor (CAR) T cells have been developed (8, 9, 10).
Recently, two BsAbs catumaxomab (11) and blinatumomab (12, 13) were approved by
the FDA, and more BsAbs directly engaging immune cells against tumor cells are now
in clinical studies. Improvements in protein engineering technology have enabled the
creation of various types of artificial antibodies with greater flexibility in design, size,
specificity, half-life and distribution, and dozens of BsAb formats have been proposed,
(6, 14).
B7 homolog 4 (B7-H4, VTCN1) is considered to be a negative regulator of immune
responses and is overexpressed in many human cancers, which indicates that B7-H4
might be a potential target for cancer therapy. B7-H4 expression is reported to be
affected by the tumor microenvironment (15, 16). Previously, we generated an
anti-human B7-H4 monoclonal antibody that induced T cell cytotoxicity to a B7-H4
positive breast cancer cell line in an in vitro indirect ADCC system, but the antibody did
not suppress the tumor growth in a mouse model (17).
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In the present study, we manufactured B7-H4/CD3 bispecific antibodies based on the
Fab and single-chain variable fragment (scFv) structure of anti-B7-H4 and anti-CD3
monoclonal antibodies using a human cell-based protein expression system. We found
that the anti-B7-H4/CD3 Fab-scFv antibody had potent cytotoxic activity against a
B7-H4-positive breast cancer cell line in vitro, and in vivo using a MHC-double
knockout (dKO) NOG mouse model (18). Here, we revealed that the anti-B7-H4/CD3
BsAb might contribute to the development of novel therapeutic antibodies against solid
tumors.
MATERIALS AND METHODS
Gene expression profile analysis
A comprehensive gene expression analysis was performed as previously described in
the HOPE (High-tech Omics-based Patient Evaluation) project at the Shizuoka Cancer
Center (19). Briefly, the tumor tissues samples were dissected from fresh surgical
specimens, and the RNA samples with an RNA integrity number (RIN) ≥6.0 were used
for the microarray analysis. The RNA was amplified, labeled, and hybridized to the
Sure Print G3 Human Gene Expression 8 × 60 K v2 Microarray (Agilent Technologies,
Santa Clara, CA, USA) and Microsoft Excel software. The data analysis was performed
using GeneSpring GX software (Agilent Technologies). Ethical approval for the study
was obtained from the institutional review board at the Shizuoka Cancer Center. Written
informed consent was obtained from all the enrolled patients. All the experiments using
clinical samples were carried out in accordance with the approved guidelines.
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Cell lines
Human breast cancer cell lines (MDA-MB-468, MDA-MB-231, ZR75, SKBR3,
HCC-1954 and HCC-1569) and the lung adenocarcinoma cell line (NCI-H2170) were
purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA),
and were maintained in RPMI1640 (SIGMA, St. Louis, MO, USA) supplemented with
10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA). Human
mammary epithelial cells (HMEC) were purchased from Lonza Ltd. (Basel,
Switzerland), and were cultured in the growth medium MEBM-CC3150 (Lonza Ltd.).
Flow cytometry and antibodies
The mouse anti-human B7-H4 antibody (clone #25) was established in-house as
previously described (17). Briefly, the human B7-H4 isoform 1 extracellular domain
was constructed and produced in the Expi293 expression system (Life Technologies
Corp.) and was immunized in BALB/cA mice. An antibody secreting hybridoma was
generated and was screened by a common method using the mouse myeloma cell line
P3X63ag8.653 (ATCC). The flow cytometric analyses were carried out on a FACS
Canto (BD Biosciences). The human mammary epithelial cells (HMECs) and cancer
cell lines were incubated with the anti-B7-H4 monoclonal Ab (clone #25) and were later
incubated with a PE-labeled polyclonal anti-mouse Ig Ab (BD Biosciences) on ice. The
following antibodies were used for flow cytometric analysis of the in vivo experiments
using humanized mice. For the human cell labeling, the anti-CD3-PerCP (HIT3a),
anti-CD4-PE or anti-CD4-PE-Cy5 (RPA-T4), anti-CD8-PE-Cy5 (HIT8a),
anti-CD11b-PE-Cy7 (ICRF44), anti-CD14-PerCP (MP9), anti-CD19-APC (HIB19),
anti-CD25-FITC (M-A251), anti-CD33-PE (WM53), anti-CD45-FITC (2D1),
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anti-CD45RA-FITC (HI100), anti-CD45RO-APC (UCHL1), anti-CD56-PE (B159), and
anti-CD127-PE-Cy7 (A019D5) were purchased from BD Pharmingen (San Jose, CA,
USA). The anti-FoxP3-PE (hFOXY) antibody was purchased from eBioscience, Inc.
(San Diego, CA, USA). The anti-TIM3-PE (F38-2E2) and anti-LAG3-FITC (17B4)
antibodies were purchased from Miltenyi Biotech (Bergisch Gladbach, Germany) and
Adipogen (San Diego, CA). The anti-mouse CD45 antibody used to label the mouse
cells was purchased from BD Pharmingen. The anti-PD-1-APC (EH12.2H7) and
anti-Ki67-PE-Cy7 (Ki-67) antibodies were purchased from BioLegend Inc., San Diego,
CA. The splenocyte and peripheral blood cells were isolated using ACK lysis buffer.
Tumor-infiltrating lymphocytes (TILs) were also separated from the control or
antibody-treated tumors by anti-human CD45-microbeads (Miltenyi Biotec) using the
autoMACS system (Miltenyi Biotec). The staining method was previously described
(16). The human cells were identified by gating human CD45+ fractions.
Production of the B7-H4/CD3 BsAb
The mouse anti-human B7H4 monoclonal antibody clone #25-derived VH and VL
genes were cloned, and construct containing Fab (B7-H4)-scFv (CD3) and scFv
(B7-H4)-scFv (CD3) linked by a (Gly4Ser)3 linker and 6× histidine-tag was designed
(Fig. 2A). These cDNAs were chemically synthesized and cloned into the expression
vector pcDNA3.3. The B7-H4/CD3 BsAbs were produced using the Expi293
expression system (Gibco, Thermo Fisher Scientific) at a ratio of 3:7 (VH-containing
long fragment:VL-containing short fragment) in the Fab-scFv format, purified with a
histidine-tag affinity column and used for experiments.
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In vitro BsAb/hPBMC cytotoxicity assay
Human peripheral blood mononuclear cells (PBMCs) were isolated from the
peripheral blood of healthy volunteers or glioma patients as effector cells using
Ficoll-Paque PLUS (GE Healthcare UK Ltd). The effector cells were incubated with
cancer cell lines or HMEC at an effector/target (E/T) ratio ranging from 1.25 to 40 in
the presence of various concentrations of Fab-scFv or scFv-scFv B7H4/CD3 BsAb at
37°C for 16 h or 24 h in a 5% CO2 atmosphere. The supernatant from the cultures were
collected and measured using a lactate dehydrogenase (LDH) cytotoxicity assay kit
(Takara Bio Inc., Seta, Shiga, Japan). The percentage of specific lysis was determined
by the following formula: percentage of specific lysis = [(effector cells and target cells
and agent release – effector cells release) – spontaneous target cell release]/(maximal
target cells release − spontaneous target cells release) × 100. The 50% effective
concentration (EC50) was calculated using a 4-parameter logistic curve fitting using
ImageJ (ver. 1.51J8, National Institutes of Health, USA). The effector T cell subsets
were isolated or depleted from the healthy volunteer PBMCs by an autoMACS
magnetic cell isolator (Miltenyi Biotec), and secreted Granzyme B and IFN- in culture
supernatants were measured by an ELISA kit (MABTECH AB, Sweden,
BioLegend,San Diego, USA).
Animal experiments
The MHC-dKO NOG mice were kindly supplied by Dr. Mamoru Ito at the Central
Institute for Experimental Animals (Kawasaki, Japan). All the animals were cared for
and treated according to the Guidelines for the welfare and use of animals in cancer
research, and the experimental procedures were approved by the Animal Care and Use
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Committee of Shizuoka Cancer Center Research Institute. The clinical experiments
using the PBMCs derived from glioma patients and healthy volunteers were approved
by the Institutional Review Board of Shizuoka Cancer Center, Shizuoka, Japan. All the
patients provided written informed consent.
In vivo imaging in the tumor-transplanted NOG-MHC dKO mice
For in vivo imaging, all the tumor-transplanted MHC-dKO NOG mice were supplied
with a low-fluorescence feed for more than one week. Cy5.5 labeling of the Fab
anti-B7-H4/CD3 antibody was performed using a Cy5.5 labeling kit (GE Healthcare
UK Ltd, Buckinghamshire England). Cy5.5-labeled Fab-scFv B7-H4/CD3 BsAb
localization was performed using the Optix MX2 laser scanner system (ART, Advanced
Research and Technologies) with an excitation at 670 nm and an emission at ≥700 nm.
The Cy5.5-labeled B7-H4/CD3 antibody was injected intravenously and imaging was
performed at sequential time points ranging from 24 h to 28 days.
BsAb pharmacokinetics in the BALB/cA mice
In the pharmacokinetic study of anti-B7-H4/CD3 BsAb, 5 nine-week-old BALB/cA
mice were injected with 100 g of Cy5.5-labeled Fab-scFv anti-B7-H4/CD3 BsAb via
tail vein, and then blood was drawn at time points ranging from 2 min to 48 h after the
antibody injection. The Serum samples were stored at -80°C until the BsAb
concentrations were measured by a sandwich ELISA or fluorescence intensity. The
sandwich ELISA was performed using the recombinant B7-H4 extracellular region and
an HRP labeled polyclonal anti-human Ig antibody (GE health care). The serum BsAb
concentration was also determined by the fluorescence intensity levels using the Optix
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MX2 imager and was performed using ART Optix Optiview software (ART, Advanced
Research and Technologies).
In vivo study using the humanized mouse model
The humanized MHC-dKO NOG mice production method was reported previously
(20). Briefly, eight-week-old MHC-dKO NOG mice were irradiated with X-rays, and 1
× 107 human PBMCs from glioma patients were intravenously administered to each
mouse via the tail vein. The study design for the experiment evaluating the mice treated
with the Fab-scFv B7H4/CD3 BsAb is shown in Fig. 5, Fig. 6 and supplementary Fig.
S5. Four in vivo experiments were performed (dose-response, short- and long-term
antitumor effect evaluation, and T-cell subset depletion). Specifically, we set the
starting day of the antibody injection as day 0. As shown in Fig. 5A (the short-term
antitumor effect experiment), on day -14, 1 × 106 MDA-MB-468 human breast cancer
cells (B7-H4 positive) or NCI-H2170 lung adenocarcinoma cells (B7-H4 negative) were
subcutaneously injected into the fat pad or flank region of the mice. Starting on day 0,
each antibody was administered intravenously. The antitumor activity was evaluated by
measuring the tumor volume. The tumor volume was calculated based on the National
Cancer Institute formula as follows: tumor volume (mm3) = length (mm) × [width
(mm)]2 × 1/2.
One week after the antibody injection, tumors, spleens, and blood were harvested
from the groups. The tumors from one set of 3 mice were used for tumor-infiltrating
lymphocyte (TIL) flow cytometry, immunohistochemistry (IHC) analysis, and real-time
qPCR of the immune response-associated genes. The schema for the long-term
antitumor effect evaluation and T cell subset depletion experiment are shown in Figs.
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5B and 5C. For the T cell subset depletion in vivo, anti-CD4 and anti-CD8 monoclonal
antibodies were purchased from Bio X Cell (West Lebanon, NH, USA).
In the dose-response experiment, as shown in Fig. 6, humanized MDA-MB-468
tumor-transplanted mice were administered BsAb sequentially in a dose escalating
manner (0.2-200 µg). Two weeks after the start of Ab injection, the tumors were
resected and used for IHC analysis.
Additional in vivo experiment targeting B7-H4-positive breast cancer cell lines, such
as HCC-1954 and HCC-1569, using the humanized MHC-dKO NOG mice was
performed in the same method as shown in supplementary Fig. S5C.
Immunohistochemistry
The xenografs were harvested one or two weeks after the injection of the
anti-B7-H4/CD3 BsAb. Formalin-fixed paraffin-embedded (FFPE) tissue blocks and
sections were made. The anti-B7-H4 antibody (clone #25 in-house made) (17),
anti-CD8 (C8/144B) and anti-CD4 (4B12) antibodies (Thermo Fisher Scientific,
Waltham, MA, USA), anti-granzyme B antibody (GrB-7, DAKO, Glostrup, Denmark),
anti-FoxP3 antibody (236A/E7, Abcam, Cambridge, MA, USA), anti-CD204 antibody
(SRA-C6, TransGenic Inc., Kobe, Japan), anti-PD-L1 antibody (28-8, Abcam) were
purchased and used for immunohistochemistry analysis. The positively stained cell
frequency was counted using the image-analyzing software, ImageJ (National Institutes
of Health, USA) in randomly selected 1/3 areas of a tumor section whole image at 200×
magnification. The necrotic area was excluded.
Human breast cancer paraffin embedded tissue arrays were purchased from US Biomax,
Inc., Rockville, MD, USA (Cat#.BR1503f) and human normal and tumor paraffin tissue
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arrays were purchased from BioChain Institute Inc., Newark, CA, USA and were used
for IHC study using anti-B7-H4 monoclonal antibody #25.
Statistical analysis
For in vivo studies, the intergroup differences were assessed by a two-way ANOVA
with a Shirley-Williams test. Significant difference in the positive cell frequency by
IHC was assessed using a two-tailed unpaired Student’s t-test. The correlation between
different gene expression levels was analyzed using a Spearman co-efficiency test. P
values of < 0.05 were considered significant.
RESULTS
B7-H4 expression in cancer tissues or cell lines
A high expression of B7-H4 was frequently observed in breast and ovarian cancer and
was partially observed in lung cancers from the HOPE project using 2527 surgically
resected tumor tissues (Fig. 1A, Supplementary Fig. S1A). The positive rate of B7-H4
mRNA expression in breast cancers (more than 2-fold upregulation in tumors compared
with normal tissues) was 56%. In contrast, PD-L1 expression was low in breast and
ovarian cancer tissues. B7-H4 mRNA expression was detected in various types of cell
lines, especially in breast cancer cell lines (Supplementary Fig. S2A) by qPCR, and
B7-H4 protein expression was detected in 43.5% of breast cancer tissues by IHC using
breast tumor tissue array (Supplementary Fig. S7). B7-H4 cell surface expression was
observed on breast cancer cells and not on HMEC (Fig. 1B, Supplementary Fig. S2C
and D).
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Unstable B7-H4 surface expression is suggested in some reports (21, 22). For example,
breast cancer cell line SKBR3 changed B7-H4 surface expression under confluent
culture conditions (Supplementary Fig. S2B).
Generation of anti-B7-H4/anti-CD3 bispecific antibodies
Two types of recombinant bispecific antibodies were constructed from the novel
anti-human B7-H4 monoclonal antibody clone #25 (17) and classical anti-CD3 antibody
(clone: OKT3). The anti-B7-H4/CD3 Fab-scFv format that connected the
antigen-binding fragment (Fab), including the human IgG4 consensus region 1, with the
single chain antigen binding fragment variable (scFv) was constructed by a single short
chain and a single long chain (Fig. 2A, B). Therefore, the 90 kDa molecular size was
larger than albumin, which contributes to the prevention of renal leakage and to the
stabilization of anti-B7-H4 affinity. The anti-B7-H4/CD3 scFv-scFv single chain
bispecific format was constructed in a manner similar to BiTE (24). The Fab-scFv
anti-B7-H4/CD3 BsAb labeled with Cy5.5 showed positive staining for B7-H4 in
positive breast cancer cell lines by flow cytometry (Fig. 2C).
PBMCs with anti-B7-H4/CD3 BsAb show cytotoxic activity against a B7-H4
positive breast cancer cell line in vitro
BsAb-mediated crosslinking of B7-H4 on the target cell surface with CD3 on T cell
causes effector T cell-dependent lysis of the target. The cytotoxic activity of the
B7-H4/CD3 BsAbs with human PBMCs against MDA-MB-468 cells for scFv-scFv was
67 ng/ml (EC50). For Fab-scFv, it was 12 ng/ml after 16 h, and for Fab-scFv it was 0.23
ng/ml after 24 h in vitro (Fig. 3A). The Fab-scFv anti-B7-H4/CD3 antibody showed
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antibody-dependent cytotoxicity on B7-H4-positive cells, whereas no cytotoxic activity
was seen against B7-H4 negative cancer cell lines (Fig. 3B, supplementary Fig. 5B).
There was no significant difference in cytotoxic activity (EC50 value) against
MDA-MB-468 cells between healthy volunteer-derived PBMCs and glioma
patient-derived PBMCs (data not shown). The anti-B7-H4/CD3 BsAb and volunteer
PBMCs showed no cytotoxic effect on a normal mammary epithelial cell line (HMEC)
(Fig. 3C). The BsAb did not kill target cells without the PBMCs (Fig. 3D, E). The
effector cell killing activity was elicited by BsAb at a dose above 1 ng/ml against
MDA-MB-468 cells after 16 h in vitro, and a decrease in the killing activity was
observed under high concentrations of BsAb (Fig. 3C, D, E), which might be caused by
a decrease in crosslinking of the target molecules because of BsAb saturation.
Unexpectedly, positively isolated CD4+ as well as CD8+ T cell subsets showed strong
cytotoxicity against B7-H4 positive tumor cell lines with granzyme B and IFN-
secretion by the stimulation of BsAb. Interestingly, even CD4 and CD8 double negative
T cells showed a weak cytotoxicity. However, granzyme B and IFN- were not
produced (Fig. 3F, Supplementary Fig. S3A, B).
Anti-B7-H4/CD3 BsAb pharmacokinetics in vivo
BsAb accumulation at the MDA-MB-468 tumor site occurred within 24 h after the
antibody injection, and the signal was detected even 28 days after the antibody injection.
In contrast, specific antibody accumulation was not recognized in B7-H4-negative
NCI-H2170 tumor (Fig. 4A and 4B, Supplementary Fig. S4). The BsAb concentration
in the serum was determined by a recombinant B7-H4 and an anti-human-IgG-Ab
sandwich ELISA, and the T1/2-beta was 8.5 h. Twenty-four hours after the 100 µg/body
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BsAb injection, the serum concentration was estimated at 0.1 g/ml (Fig. 4C), but the
serum concentration by fluorescent imaging of the Cy5.5-labeled BsAb was
approximately 1 g/ml after 24 h (Supplementary Fig. S4), which was higher than the
antibody level obtained by the sandwich ELISA method. The BsAb in the sera may
partially exist in complex with other proteins.
Antitumor effect of the anti-B7-H4/CD3 BsAb against MDA-MB-468 and other
breast tumors
A single injection of Fab-scFv B7-H4/CD3 BsAb, triple-negative breast cancer
MDA-MB468 xenograft tumors decreased in size by about 70% in hPBMC-
transplanted humanized NOG mouse model in a week (Fig. 5A). In the long-term
experiment, the reduction in tumor size was maintained until 2 weeks after the BsAb
injection (Fig. 5B). In contrast, the growth of the tumors treated with the full-body
anti-B7-H4 antibody (clone #25) was not inhibited. Additionally, no antitumor effect
was observed in B7-H4-negative NCI-H2170 tumors treated with BsAb (Fig. 5A). The
BsAb also induced T cell cytotoxicity to HER2 positive and B7-H4 positive breast
cancer cell lines, HCC-1954 and HCC-1569 in vitro (Supplementary Fig. S5A, B), and
BsAb injection suppressed HCC-1954 and HCC-1956 tumor growth by more than 50%
in the humanized mouse model (Supplementary Fig. S5C).
A relatively high dose of anti-CD3 antibody (clone: OKT3), formerly used in the clinic
as an immunosuppressive agent, resulted in no inhibitory effect and obvious weight loss.
The BsAb-treated mice showed no adverse effects such as weight loss. An escalating
BsAb dose (0.2-200 µg/body) was administered without harm (Fig. 6A).
In the T cell subset depletion in vivo study, CD8+ T cell depletion blocked the growth
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inhibition induced by B7-H4 BsAb, and CD4+ T cell depletion showed only a weak
blocking effect on growth inhibition (Fig. 5C). These results demonstrated that the
antitumor effect of anti-B7-H4 BsAb was mediated partially by CD4+ T cells and
mostly by CD8+ T cells.
Effector immune cells analysis of MDA-MB468 tumor-bearing mice treated with
the anti-B7-H4/CD3 BsAb
From the flow cytometry analysis of TIL and splenocytes from the antibody-treated
mice, the total cell number and CD3+CD45+ T cell number from spleens showed a
tendency to increase in the BsAb-treated mice compared with the controls
(Supplementary Table 1), but there were no significant differences in the TIL T cell
subset (data not shown).
In the Fab-scFv anti-B7-H4/CD3 BsAb-administered group, hematoxylin-eosin (HE)-
stained tumor specimens showed remarkable infiltration of lymphoid cells inside the
tumor cores and resulted in almost no viable cancer cells at the tumor site (Fig. 6B).
Immunostaining revealed that CD8+ and granzyme B+ lymphocytes were more
frequently observed in the BsAb-treated group (Fig. 6B, C), but CD4+ and FoxP3+
lymphocytes and CD204+ immune cells did not significantly differ (data not shown).
IHC study of B7-H4 in human normal and tumor tissue arrays
In 28 human normal and tumor tissues array analysis, seven B7-H4-positive tumors
(pharynx, esophagus, stomach, lung, kidney, fallopian tube and kidney) were identified
(Supplementary Fig. S6). In the normal tissues, only the tonsil (epithelial cell) was
positively stained with the anti-B7-H4 antibody clone #25. The B7-H4 IHC study of
16
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human breast cancer tissue array showed that a B7H4-positive rate of 69 breast cancer
tissues was 43.5%, and there was a tendency of high B7H4 stain in triple negative tumor
compared with PD-L1 (Supplementary Fig. S7). However, it was not definite because of
small number of cases.
DISCUSSION
Because of the recent success of immune checkpoint blocking antibodies, such as
ipilimumab and nivolumab, in patients with metastatic melanoma and other
malignancies, clinical trials are underway to evaluate their efficacy in various solid
cancers (1-4). These studies show that the modulation of the suppressed immune system
is an effective way to combat cancers and that immunotherapy can be used to treat
cancer.
In addition to immune checkpoint antibodies, bispecific antibodies that directly
engage immune cells (25) are becoming another promising strategy in cancer antibody
therapy. Advances in recombinant protein technology allow for the construction of
bispecific antibodies in a variety of formats with great flexibility. A number of formats
have been proposed including bispecific T cell engager (BiTE) (26, 27), tandem
diabody (28), dual-affinity-retargeting (DART) (29), and antibody-TCR format
(ImmTac) (30). Catumaxomab targeting EpCAM and Blinatumomab targeting CD19
are approved for clinical use and others are in various stages of clinical development
(31-34).
New T cell-engaging bispecific antibodies, including BiTE and DART, work at a much
lower dose in xenograft models and clinical use (24, 29) compared to conventional
17
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antibody therapies. Our BsAb, which connects the anti-B7-H4 Ab and anti-CD3 Ab,
immediately elicited a strong B7-H4 positive target tumor lysis at very low dose by
CD8+ effector T cells or other T cell subsets, bypassing peptide-MHC/TCR recognition
(Figs. 3 and 5).
Trastuzumab and other HER2-targeted agents improved patient outcomes in breast
cancer therapy, but initially the responsive tumors develop resistance. HER2-targeted
BsAb showed an antitumor potency against trastuzumab-resistant tumor cells in in vitro
and in vivo models (33). Its effective concentrations (EC50) was inversely correlated
with surface HER2 expression and could be effective for tumors that express a lower
level of HER2. Given that B7-H4 is frequently expressed on the breast cancer cells
irrespective of HER2 expression (Supplementary Fig. S8) and that B7-H4 expression
has a tendency to be higher in triple negative breast cancer cells (35), B7-H4 is
potentially a novel substitute target for primary and recurrent breast cancers, including
triple negative cancers that are nonresponsive to HER2 targeting therapies. Interestingly,
our anti-B7-H4 BsAb showed potent anti-tumor effect in vivo against B7-H4-positive
breast cancer cell lines, HCC-1954 and HCC-1569, which was demonstrated to be basal
type-HER2-positive and trastuzumab-resistant by some researchers (36, 37). This
observation might suggest that this type of breast cancers with positive B7-H4
expression could be the target for B7-H4 targeting therapy.
Additionally, based on B7-H4 gene expression and the clinical data from the TCGA
tumors samples, high B7-H4 gene expression can be a poor prognostic factor in breast
cancer patients (Supplementary Fig. S1B).
B7-H4 is widely detected in cancers and normal tissues at the mRNA level, but its cell
surface expression is limited and tends to be unstable (21, 22). Our IHC study showed a
18
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limited expression in normal tissues and positive expressions in several tumor tissues
other than breast cancer (Supplementary Fig. S6).
The BsAb eliminated breast cancer cells in a short amount of time (Fig. 5A, 6B), and
the immediate antitumor response may help overcome cancers with continuous genomic
evolution and immune evasions in contrast to the immune checkpoint blockade antibody
therapy, which takes several weeks to induce an adequate amount of tumor antigen
specific effector T cells at the tumor site.
Tumor specific antigens, such as carcinoembryonic antigens, cancer/testis antigens or
differentiation antigens, are also expressed in normal tissues to varying degrees, except
for mutation neoantigens. Therefore, the therapeutic approaches targeting these antigens
requires antibody dose control for harnessing cytotoxic effector cells. B7-H4-targeted
CAR-T cell treatment showed lethal toxicity including a delayed
graft-versus-host-disease (GVHD) in a mouse model (23), and a clinical trial using
trastuzumab-based CAR-T cells resulted in patient death and was aborted (38). These
reported adverse events suggest that the strict regulation of effector function is
important for clinical use. A shorter in vivo half-life of the smaller size BsAb is
described as a negative property, but this characteristic enables easier control of effector
cells for clinical use compared with CAR-T cell therapy.
Immune check point modulators are not effective for the majority of cancer patients,
and positive outcome is likely to require a high mutation burden and effector T cell
accumulation against mutation-derived neoantigens (39, 40). BsAb therapy may be an
alternative anticancer immunotherapeutic strategy to bypass neoantigen-MHC/TCR
recognition.
19
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In this study, we demonstrated that the B7-H4-targeted bispecific antibody had potent
antitumor activity in vitro and in vivo, which was specific for B7-H4-positive tumors
and was not cytotoxic on a normal mammary duct epithelial cell line. More importantly,
in vivo imaging showed a long-lasting retention of the injected antibody at the tumor
site. These results suggest that the Fab-scFv anti-B7-H4/CD3 bispecific antibody might
be a therapeutic agent for PD-L1-negative B7-H4-expressiong tumors or anti-HER2
antibody-non-responsive breast tumors.
Acknowledgements
We thank Mr. Koji Takahashi for his excellent assistance in maintaining the
NOG-dKO mouse in the animal facility. This study was supported by JSPS KAKENHI,
Japan (Grant No. 17K07235; to A. Iizuka).
Abbreviations: B7-H4, B7 homolog 4; BsAb, bispecific antibody; PBMC, peripheral
blood mononuclear cell; MHC, major histocompatibility complex; NOG,
NOD/Shi-scid-IL-2Rnull; GVHD, graft versus host disease; PD-1, programmed death-1;
PD-L1, programmed death-ligand-1; HLA, human leukocyte antigen; HER2, human
EGFR-related 2; IHC, immunohistochemical
Author contributions:
Yasuto Akiyama designed the study, drafted the manuscript, and were responsible for
completing the study. Akira Iizuka and Tadashi Ashizawa performed in vivo
experiments and were responsible for all animal studies. Akira Iizuka, Chizu Nonomura,
Keiichi Ohshima and Ryota Kondo participated in the designing the experiments and
20
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performed the biological assays and bioinformatics analysis. Takashi Sugino examined
and diagnosed the pathological sections derived from the mice. Koichi Mitsuya,
Nakamasa Hayashi and Yoko Nakasu were responsible for supplying the
patient-derived materials for the clinical research. Kouji Maruyama was involved in
maintaining the NOG-MHC dKO mouse in the animal facility. Ken Yamaguchi,
reviewed the manuscript. All the authors read and approved the final draft.
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Figure legends
Figure 1. Expression of the B7-H4 gene in breast and gynecological cancer tissues. (A)
A comprehensive DNA microarray analysis was performed using 2527 surgically
resected cancer tissue samples and the major cancer categories are indicated. The box
plots of log2-normalized values of VTCN1 (B7-H4) expression are displayed. The data
analysis was performed using Microsoft Excel 2016 software. (B) The B7-H4
expression in the human mammary epithelial cells (HMECs) and various cancer cell
lines was determined by flow cytometry. An in-house anti-B7-H4 monoclonal Ab (clone
#25) was used.
Figure 2. Design and production of the anti-B7-H4/CD3 bispecific antibodies in
Fab-scFv and scFv-scFv formats. (A) Molecular design of the BsAbs; Fab
(anti-B7-H4)-scFv (anti-CD3) construct consisting of mouse anti-human B7H4
monoclonal antibody clone #25-derived VH, and VL genes and human immunoglobulin
constant region domain sequences linked by (Gly4Ser)3 linker sequence to the
anti-human CD3 antibody VH and VL genes. (B) Coomassie blue-stained SDS-PAGE
of purified anti-B7-H4/CD3 bispecific antibodies, Fab-scFv containing a single short
chain (30 kDa) and single long chain (60 kDa) and sc-Fv-scFv containing a single chain
(60 kDa). (C) Characterization of the Fab-scFv anti-B7-H4/CD3 bispecific antibody.
The binding activity of Cy5.5-labeled Fab-scFv anti-B7-H4/CD3 BsAb to the B7-H4
(+) breast cancer cell line was evaluated by flow cytometry. Gray dotted line: no
antibody, black thick line: Cy5.5-labeled anti-B7-H4/CD3 BsAb.
Figure 3. Cytotoxic activity of the anti-B7-H4/CD3 BsAb against breast cancer cell
27
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lines. (A) The 50% effective concentration (EC50) of each BsAb for cytotoxic activity
against breast cancer MDA-MB-468 cells is shown. Open circle: scFv-scFv 67 ng/ml,
closed circle: Fab-scFv 13 ng/ml, and closed triangle: Fab-scFv 0.2 ng/ml. Effector
human PBMCs were derived from one representative case of three healthy volunteers
and were used at an effector/target ratio (E/T) 40. Human PBMCs from a glioma patient,
which were also used for in vivo experiment, were used for a cytotoxicity assay (data
not shown). The EC50 values were calculated using ImageJ. (B) Cytotoxic activity of
anti-B7-H4/CD3 BsAb against B7-H4-positive or negative cancer cell lines. The E/T
ratio was set at 40. Square: ZR75, cross: MDA-MB-231, triangle: SKBR3, diamond:
NCI-H2170. (C) Cytotoxic effect of the anti-B7-H4/CD3 BsAb against HMECs.
PBMCs derived from 3 different healthy volunteers were used as effector cells. Closed
marker: MDA-MB-468, open marker: HMEC. Each point shows the average of 2
experiments from one volunteer. The cytotoxic activity of anti-B7/CD3 BsAb in various
E/T ratios using (D) Fab-scFv and (E) scFv-scFv against MDA-MB-468. From the top
line, E/T ratios of 20, 10, 5, 2.5, 1.25, and target cells alone are shown. (F) Cytotoxicity
assay against MDA-MB-468 using positively or negatively MACS-isolated T cell
subsets. The data is representative of three independent experiments with each volunteer
T cell subsets at an E/T of 10 for 16 h.
Figure 4. Cy5.5-labeled anti-B7-H4 BsAb (Fab-scFv) localization in tumor-bearing
mice and BsAb half-life in mouse blood. (A) Sequential fluorescence imaging of the
MDA-MB-468 tumor-bearing MHC-dKO NOG mouse after Cy5.5-labeled BsAb
intravenous injection from 24 h to 28 days. (B) Fluorescence imaging of the
NCI-H2170 tumor-bearing MHC-dKO NOG mouse at 24 h after the BsAb injection.
28
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(C) Fluorescence imaging in the resected organs including the MDA-MB-468 tumor on
day 28 after Cy5.5-labeled BsAb injection (200 g/body). (D) Serum BsAb
concentration after a 100 g injection into 5 BALB/cA mice. The blood was
sequentially collected from 2 min to 48 h after the injection to measure serum BsAb
concentration. Each point represents the mean ± SD of 5 mice.
Figure 5. Antitumor effect of the anti-B7-H4/CD3 bispecific antibody in vivo using a
humanized MHC-dKO NOG mouse model. (A) The short-term antitumor effect of the
antibodies on the MDA-MB-468 tumors. Two weeks after the transplantation of
PBMCs and cancer cells, each antibody was administered by a 40 µg/body single
injection via the tail vein. Diamond: control group, closed square: Fab-scFv
anti-B7-H4/CD3 BsAb, triangle: anti-B7-H4 mouse monoclonal antibody (mAb) (clone
#25), and cross: anti-human CD3 mAb (OKT3). *P < 0.05. (B) The long-term antitumor
effect of the antibodies on the MDA-MB-468 tumors. Twelve days after the
transplantation of PBMCs and cancer cells, the BsAb was administered at 40 µg/body
via the tail vein two times weekly. Diamond: control group, closed square: Fab-scFv
anti-B7-H4/CD3 BsAb. Each point shows the mean ± SD value of 7 mice. *P < 0.05.
(C) The effect of T cell depletion on the antitumor effect of anti-B7-H4/CD3 BsAb.
Human T cell subsets were depleted by i.p. injection of 100 g/body/day anti-CD4 or
anti-CD8 mAb (Bio X Cell) from day 7 to day 9. Diamond: control group (no antibody),
closed square: isotype antibody + BsAb, closed circle: anti-CD4 antibody + BsAb, and
closed triangle: anti-CD8 antibody + BsAb. Each point shows the mean ± SE value of 4
mice.
29
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Figure 6. Breast cancer cell eradication and effector T-cell infiltration inside the tumor
after BsAb treatment. (A) PBMCs and MDA-MB-468 tumor-transplanted mice were
administered BsAb sequentially in a dose escalating manner (0.2-200 µg) with a 3 or 4
day intervals between injections. Two weeks after the initial Ab injection, the resected
control tumor specimens and the BsAb treated-tumor specimens with about 70% size
reduction were used for IHC analysis. (B) Images of the anti-B7-H4 BsAb treated
mouse tumors stained with hematoxylin-eosin (HE) or with anti-B7-H4 (clone #25) and
anti-CD8 antibodies. Magnification: 200×. (C) Infiltrating CD8+ or granzyme B+ T cell
counts at the tumor site. Each histogram represents the mean ± SD of more than 10
areas of the tumor section. *P < 0.05.
30
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Fig. 1
A
expression (log2) VTCN1 (B7-H4) gene
Breast Gynecologic Lung Gastric Hepatic Neural Colorectal (n: 159) (n: 81) (n: 439) (n: 303) (n: 143) (n: 48) (n: 760) B MDA-MB-468 ZR75 MDA-MB-231 SKBR3 100 80 60 40
% of Max 20 0 102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105 Human mammary NCI-H2170 (Lung) HCC1954 HCC1569 epithelial cell (HMEC)
2 3 4 5 102 103 104 105 10 10 10 10 102 103 104 105 102 103 104 105 Anti-B7-H4 Ab
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Fig. 2
A Fab-scFv B (26kDa + 54kDa) MW.(Da) N 200k Anti-B7H4-VL CL C Anti-CD3-VH SS 116k N 97k Anti-B7H4-VH IgG4-CH1 Anti-CD3-VL Tag-C 66k
scFv-scFv 45k (59kDa) 31k N Anti-B7H4-VL Anti-CD3-VH 21.5k Anti-B7H4-VH Anti-CD3-VL Tag-C 14.4k 6.5k
C MDA-MB-468 ZR75 SKBR3 MDA-MB-231 100 80 60 40 % of Max 20 0 102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105 Cy5.5-Fab-scFv
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Fig. 3
A EC50 : ▲ Fab-scFv (24H): 0.23 ng/ml D 50
● Fab-scFv (16H): 12 ng/ml 40
○ scFv-scFv (16H): 67 ng/ml 30 70 20 60 Lysis (%) Lysis 10 50 40 0
(%) 30 -10 1 2 3 4 5 20 0 10 10 10 10 10 10 Fab-scFv (ng/ml) Target cells lysis lysis cells Target 0 E 50 -10 40 -1 0 1 2 3 0.010 0.110 110 1010 10010 100010 BsAb conc. (ng/ml) 30 20
B 70 (%) Lysis 10 60 50 0 40 -10 1 2 3 4 5 30 0 10 10 10 10 10 20 scFv-scFv (ng/ml) Lysis (%) Lysis 10 100 0 F BsAb: 0 ng/ml -10 80 BsAb: 10 ng/ml 0.10 1010 10101 100102 1000103 Fab-scFv (ng/ml) 60
50 40 C (%) Lysis 40 20 30
20 0
10 -20 Lysis (%) Lysis Whole CD4(-) CD8(-)
0 CD4(+) CD8(+)
-10 0 10-2 10-1 100 101 102 103 104 105 CD4(-)CD8(-) Whole/no target Fab-scFv (ng/ml)
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Fig. 4
A Cy5.5-Fab-scFv iv.: 5μg/body B Cy5.5-Fab-scFv iv.: 10μg/body
MDA-MB-468 24 h 3 days 7 days MDA-MB-468 Pre 24 h 100
Tumor Tumor 0
Pre injection 14 days 21 days 28 days NCI-H2170 Pre 24 h 120 100
0 0
C Cy5.5-Fab-scFv iv.:200μg/body D 1000 28 days 154 Lung 100 (T : 8.5 hours) Tumor 1/2-β 10 Heart 1 (µg/ml) Liver BsAb/ serum 0.1
0.01 Kidney 0.001 Spleen 0 0 10 20 30 40 50 60 Hours
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Fig. 5
A B -13d -12d 0d 7d 14d (Study scheme) MHC-KO NOG mice X-ray, MDA-MB-468 BsAb BsAb -15d -14d 0d 7d hPBMC iv. sc. iv. iv.
350 2.5Gy X-ray Cancer Antibody Sampling 300 Human cell line injection 1. Tumor; qPCR, TIL-FCM
) 250 PBMC iv. sc. iv. 2. Spleen; qPCR, FCM 3 3. Blood; qPCR 200 (mm 150 * 100 * MDA-MB-468 Tumor volume * 50 *
) 200
3 Control 0 -5 0 5 10 15 150 BsAb Days after BsAb injection 100 Anti-B7H4 Ab (#25) C 50 Anti-CD3 Ab -13d -12d -5d 0d 7d 14d * (OKT3) Tumor Volume (mm 0 -2 0 2 4 6 8 Validating Days after BsAb injection X-ray, MDA- Dep-Ab BsAb BsAb hPBMC MB-468 ip. 3 days iv. iv. depletion iv. sc. NCI-H2170 350 )
3 800 Control 300 600 250 BsAb )
3 200 400 150 (mm 200 100 Tumor volume 50 Tumor Volume (mm 0 -2 0 2 4 6 8 10 12 14 0 Days after BsAb injection -5 0 5 10 15 Days after BsAb injection
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Fig. 6
A 2.5Gy X-ray C BsAb iv. (/body) 2000 300
hPBMC iv. Tumor ) 2
-16d sampling ) 250 * for IHC 2 * MDA-MB-468 sc. 0d 3d 6d 10d 14d 1500 -15d 200
300 1000 150
Cont. ) 3 200 BsAb 100
CD8+ cells(/mm 500
(mm 50 100 Granzyme Granzyme B+ cells (/mm Tumor volume 0 0 0 Control BsAb -16-14-12-10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 Control BsAb Days after BsAb injection (*p < 0.005)
B HE B7-H4 CD8
Control
BsAb treated
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A T-cell-engaging B7-H4/CD3 bispecific Fab-scFv antibody targets human breast cancer
Akira Iizuka, Chizu Nonomura, Tadashi Ashizawa, et al.
Clin Cancer Res Published OnlineFirst February 8, 2019.
Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-3123
Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2019/02/08/1078-0432.CCR-17-3123.DC1
Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.
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