This clinical research project using comprehensive whole exome sequencing and gene expression profiling of various tumor tissues, called the High-tech Omics-based Patient Evaluation (HOPE) for Cancer Therapy, has been conducted in accordance with the ʻEthical Guidelines for Human Genome and Genetic Analysis Researchʼ, revised in 2013. Informed consents were obtained from all patients participating in the HOPE project, which was approved by the Institutional Review Board of the Shizuoka Cancer Center (SCC), Japan. Tumor tissues, along with surrounding normal tissues, were dissected from surgical specimens by trained pathologists.

BALB/cA mice and nude mice (BALB/cA-nu/nu) were obtained from Nippon Clea (Tokyo, Japan). All animals were cared for and used humanely according to the Guidelines for the Welfare and Use of Animals in Cancer Research (25). All procedures were approved by the Animal Care and Use Committee of the Shizuoka Cancer Center (SCC) Research Institute.

Total RNA was extracted from ~10 mg of tissue samples using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Following assessment using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), total RNA with an RNA integrity number (RIN) of six or higher was used for DNA microarray analysis. Gene expression analysis was performed using SurePrint G3 Human GE 8×60K v2.0 arrays (Agilent Technologies) according to the manufacturer's instructions. Signal data analysis was carried out using GeneSpring version 13.1.1 software (Agilent Technologies). The ratio of tumor tissue vs. surrounding non-cancerous tissue was calculated from the normalized values.

The human B7-H4 isoform 1 extracellular domain was constructed with a 6X histidine tag in a pcDNA3.3 expression vector, and the B7-H4 extracellular soluble free form was harvested from Expi293F™ cell (Life Technologies Corporation) culture supernatant, affinity-purified, and used for experiments. B7-H1 and B7-DC were produced in the same way for use as negative controls. After the immunization of BALB/cA mice, an antibody secreting hybridoma was generated by a common method using the mouse myeloma cell line P3X63Ag8.653 (American Type Culture Collection; ATCC, Manassas, VA, USA).

Specificity was validated by sandwich ELISA and fluorescent staining of B7-H4-transfected HEK293 cells. Briefly, purified newly-generated antibodies were immobilized on a 96-well microplate Nunc Immobilizer Amino Surface (Thermo Fisher Scientific, Inc.) prior to the addition of 3% bovine serum albumin for overnight blocking and 10 µg/ml B7-H4 (soluble form) or controls. After being washed, 2 µg/ml biotinylated antibody (Ab) clone #25 was added and detected by HRP-conjugated streptavidin (Thermo Fisher Scientific, Inc.).

HEK293 cells that transformed with the full B7-H4 sequence or cancer cell lines were incubated with 8 µg/ml Ab clones and later incubated with 4 µg/ml PE-labeled polyclonal anti-mouse Ig Ab (BD Biosciences) on ice. Fluorescence intensity was determined by the flow cytometer FACSCanto (BD Biosciences).

Surface plasmon resonance (SPR) analysis was performed on a Biacore ×100 (GE Healthcare) in order to determine the kinetics of the anti-B7-H4 Ab and free form of B7-H4. All reagents and sensor chips were purchased from GE Healthcare. Immobilization of anti-B7-H4 IgG antibodies on the CM5 sensor chip was performed at pH 5.0, and the Ab binding was targeted to 1000 response units (RU). To regenerate the sensor chip, 10 mM glycine-HCl pH 1.7 was used. The binding kinetics were monitored by injecting multiple concentrations of B7-H4 in HBS buffer (10 mM HEPES pH 7.4, containing 0.15 M NaCl, 3 mM EDTA, and 0.05% Tween-20) at a flow rate of 30 µl/min at 25°C. The dissociation phase was also monitored by HBS buffer flow. The binding kinetic parameters were calculated by Biacore ×100 evaluation software.

Breast cancer cell line lysates harvested with Laemmli sample buffer, and over-confluent culture supernatants were used for western blot analysis. Cell lysates and supernatants were electrophoresed through a SDS-PAGE gradient gel and transferred to a PVDF membrane. After overnight blocking with 5% skim milk and washing with 0.05% Tween-20-PBS (T-PBS), the membrane was incubated for 1 h at room temperature (RT) with rabbit anti-human B7-H4 Ab (clone, EP1165). After being washed with T-PBS, the membrane was incubated with a secondary Ab, HRP-conjugated donkey anti-rabbit IgG polyclonal Ab (GE Healthcare) diluted 1:10,000. ECL Plus (GE Healthcare) was used for detection according to the manufacturer's instructions.

Female nude mice (BALB/cA-nu/nu, 5–6 weeks old) were transplanted subcutaneously with human breast cancer MDA-MB-468 or lung cancer NCI-H2170 cells. Formalin-fixed paraffin-embedded (FFPE) cancer tissue blocks were made. To evaluate B7-H4 expression, sections from the cancers were immunostained with anti-B7-H4 Ab (clone #25) or mouse IgG1 isotype control (BD Pharmingen) and later stained with hematoxylin.

MDA-MB-468 cells (1×10⁷) were inoculated into the mammary fat pad of BALB/cA-nu/nu mice. Antibody clone #25, at doses of 2 or 10 mg/kg, was administered by intraperitoneal injection twice a week from day 9 to day 27 after tumor inoculation. To evaluate the antitumor activity against inoculated tumors, tumor volume was calculated based on the National Cancer Institute formula as follows: Tumor volume (mm³) = length (mm) × [width (mm)]² × 1/2.

Briefly, effector cells were isolated from healthy volunteer peripheral blood by Ficoll-Paque PLUS (GE Healthcare Life Sciences, Buckinghamshire, UK) and then enriched by depletion of CD14⁺ or CD19⁺ cells using MicroBeads and autoMACS (Miltenyi Biotec K.K., Tokyo, Japan). The effector cells were incubated on ice for 10 min with 0.2 mg/ml (high concentration) anti-CD3 Ab (OKT3) diluted with staining buffer (0.5% BSA, 0.1% azide, 1 mM glucose-PBS), followed by washing. Cells were then incubated with 0.5 mg/ml polyclonal rabbit anti-mouse immunoglobulins (DakoCytomation, Glostrup, Denmark) and after being washed, they were incubated with 0.5 mg/ml of each anti-B7-H4 antibody. The effector cells were washed and resuspended to a concentration of ~10⁷ cells/ml in 10% FBS-RPMI-1640 and co-cultured with non-radioactive reagent-labeled MDA-MB-468 target cells in a 96-well round-bottom plate at 37°C for 3 h. Target cell lysis was performed with DELFIA^® EuTDA Cytotoxicity Assay Reagents and measured using a Wallac 1420 ARVO SX multilabel counter (PerkinElmer, Waltham, MA, USA). The percentage of specific lysis was determined by the following formula: Percentage of specific lysis = (experimental release − spontaneous release)/(maximal release − spontaneous release) × 100.

Statistically differences were analyzed using the Student's paired two-tailed t-test. Values of P<0.01 were considered to indicate statistically significant differences.

We found that B7-H4 expression was upregulated in some types of cancer when compared to expression in non-cancerous normal tissues (Fig. 1). B7-H4 expression was upregulated in breast, lung, and gynecological cancers and downregulated in skin and urinary tract cancers. Expression of other genes from the B7 family was not apparently altered. B7-H4 expression was detected in breast and ovarian cancer cell lines by RT-PCR in accordance with the previous study (data not shown), but the soluble free form of B7-H4 in the culture medium was not, as determined by ELISA. The splice variants of B7-H4 that were detected corresponded mainly to isoforms 1, 2, and 4 and partially to isoform 3 which has a different sequence.

We generated 19 hybridoma clones of anti-human B7-H4 isoform 1 for detection of the natural form, and purified eight antibody clones which could be cultured in vitro under serum-free conditions. The antibody clones bound B7-H4 but not B7 homologs PD-L1 or PD-L2 (B7-DC) (Fig. 2A), and the staining of HEK293 cells transfected with the full B7-H4 isoform 1 sequence showed two different types of staining, strong (#18, #25) and weak (#113, #121, #209), by flow cytometry. All mAb clones were confirmed to have different DNA sequences (Fig. 2B).

Sandwich ELISAs using antibody clone #25 as both the capture and detector antibody detected the free form of B7-H4; ELISA using clone #18 as the capture Ab showed a similar curve. These two capture Abs had lower sensitivities than the other clones, which contrasted to staining of HEK293 transfectants (Figs. 3A and 2B). B7-H4 is a monovalent molecule, and thus these observations were indicative of B7-H4 homodimerization and bivalent antigenicity. In determining the affinity kinetics of the antibodies to the free form of B7-H4, both clone #25 and the commercially available clone-H74 showed two-state reaction curves, including rapid dissociation and stable dissociation curves, which can be explained by dimer dissociation and monomer dissociation, respectively (Fig. 3B). Each rapid dissociation rate was equivalent.

B7-H4 expression in the breast cancer cell lines was validated by western blot analysis. Predicted molecular weight of B7-H4 is 29 kDa but it aligns with a size of 50~70 kDa owing to glycosylation and isoform variations (14,26). B7-H4 was detected in cell lysates but not in culture supernatants (Fig. 4A). The soluble-free forms have reportedly been detected in patient sera, but it is difficult to detect in vitro in culture supernatant.

We compared B7-H4 staining in the MDA-MB 468 cell line under suppressive (azide-containing at 4°C) or normal (azide-free at RT) conditions by flow cytometry (Fig. 4B). B7-H4 staining was positive under suppressive conditions using clone #18 and clone #25 antibodies; however, there was no detection under normal conditions. In addition, B7-H4 staining was also not apparent in samples that underwent an overnight incubation at 37°C and 5% CO₂.

In the MDA-MB-468 transplanted nude mouse model, the strong B7-H4 expression on the tumor cell membrane was observed by immunohistochemistry (IHC), and the intensity of the staining was heterogeneous in the tumor tissue (Fig. 5A). By contrast, the B7-H4-negative NCI-H2170 cell line (control) did not show any B7-H4 expression in tumor tissues. We administered antibody clone #25 (mouse IgG1) to the MDA-MB-468 transplanted mouse model, but no tumor suppressive effect was observed (Fig. 5B).

Based on the observations that i) there was loss of B7-H4 expression on the tumor cell surfaces under usual conditions and ii) there was lack of antitumor activity due to non-functional ADCC in vivo, we employed an indirect ADCC-redirecting T-cell cytotoxicity assay to study B7-H4 using polyclonal anti-mouse IgG-mediated linking of anti-CD3 and anti-B7-H4 antibodies. Freshly enriched T cells indirectly targeting B7-H4 exhibited clone #25-dependent cellular cytotoxicity against the MDA-MB468 cells (Fig. 6). By contrast, clones #18 (mIgG2b), #113 (mIgG1) and #209 (mIgG1) did not induce targeted cell death.