Oral squamous carcinoma cell lines including Ca9-22 (derived from gingiva), HO-1-u-1 (mouth floor), HSC-2 (oral cavity), and SAS (tongue) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). LN229 (glioblastoma cell line), MDA-MB-468 (breast cancer), BT-474 (breast cancer), and P3U1 (mouse myeloma) were obtained from the American Type Culture Collection. LN229/HER2 cells were established in a previous study (14). P3U1 cells were cultured in RPMI-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan). LN229, LN229/HER2, MDA-MB-468, BT-474, Ca9-22, HO-1-u-1, HSC-2, and SAS were cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc.), 100 units/ml of penicillin, 100 µg/ml streptomycin, and 25 µg/ml amphotericin B (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂.

All animal experiments were performed in accordance with relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal experiments for hybridoma production were approved by the Animal Care and Use Committee of Tohoku University (permit no. 2016MdA-153). Animal health was monitored daily. Animal studies for Antibody-Dependent Cellular Cytotoxicity were approved by the institutional committee for experiments of the Institute of Microbial Chemistry (permit no. 2019-066). Animal studies for antitumor activity were approved by the institutional committee for experiments of the Institute of Microbial Chemistry (permit no. 2019-014). Mice were monitored for health and weight every 3 or 4 days. Experiment duration was three weeks. A bodyweight loss exceeding 25% and a maximum tumor size exceeding 3,000 mm³ were identified as humane endpoints. Mice were euthanized by cervical dislocation, and the death was verified by respiratory arrest and cardiac arrest.

One four-week-old female BALB/c mouse was purchased from CLEA Japan and housed under specific pathogen-free conditions. Anti-HER2 hybridoma cells were produced as described previously (14). Briefly, the BALB/c animal was immunized by intraperitoneal (i.p.) administration of 100 µg recombinant HER2 extracellular domain along with Imject Alum (Thermo Fisher Scientific Inc.). After several additional immunizations, a booster dose was administered i.p. 2 days before harvesting spleen cells. Mice were euthanized by cervical dislocation, and the death was verified by respiratory arrest and cardiac arrest. Spleen cells were then fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN, USA). The resulting hybridoma cells were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine selection medium (Thermo Fisher Scientific, Inc.). Culture supernatants were screened using enzyme-linked immunosorbent assays with recombinant HER2 extracellular domain. mAbs were purified from the supernatants of hybridoma cells and cultured in Hybridoma-SFM medium (Thermo Fisher Scientific, Inc.) using Protein G Sepharose 4 Fast Flow (GE Healthcare UK Ltd.).

Hybridoma cells were harvested by brief exposure to 0.25% trypsin/1-mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin in phosphate-buffered saline (PBS), cells were treated with 1 µg/ml anti-HER2 (H₂Mab-19) for 30 min at 4°C and subsequently with Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000; Cell Signaling Technology, Inc.). Fluorescence microscopy data were collected using an EC800 Cell Analyzer (Sony Corp.).

Histologic sections (catalog no. T8235721-5; lot no. B104066; BioChain Institute Inc.) were purchased in this study. Four-µm histologic sections from paraffin blocks of resected xenografts were also produced. These sections were deparaffinized in xylene, then rehydrated and autoclaved in citrate buffer (pH 6.0; Agilent Technologies Inc.) for 20 min. Sections were incubated with primary mAbs for 1 h at room temperature, then treated using an Envision+ kit (Agilent Technologies Inc.) for 30 min. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (Agilent Technologies Inc.) for 2 min, and sections were then counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

Histologic sections (catalog no. T6235086-1, BioChain Institute Inc.) were incubated with 1 µg/ml of primary mAbs for 1 h at room temperature and were then treated using an Envision+ kit (Agilent Technologies Inc.) for 30 min. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (Agilent Technologies Inc.) for 2 min, and sections were then counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

Cells were suspended in 100 µl serially diluted H₂Mab-19 (6 ng/ml-100 µg/ml), followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.). Fluorescence microscopy data were collected using an EC800 Cell Analyzer (Sony Corp.). The dissociation constant (K_D) was obtained by fitting binding isotherms to built-in one-site binding models in GraphPad PRISM 6 (GraphPad Software, Inc.).

Six six-week-old female BALB/c nude mice were purchased from Charles River. After euthanization by cervical dislocation, spleens were removed aseptically and single-cell suspensions obtained by forcing spleen tissues through a stainless steel mesh using a syringe. Erythrocytes were lysed with a 10-sec exposure to ice-cold distilled water. Splenocytes were washed with DMEM and resuspended in DMEM with 10% FBS and used as effector cells. Target cells were labeled with 10-µg/ml Calcein AM (Thermo Fisher Scientific, Inc.) and resuspended in the same medium. The target cells (2x10⁴ cells/well) were plated in 96-well plates and mixed with effector cells, anti-HER2 antibodies, or control IgG (mouse IgG_2b) (Sigma-Aldrich Corp.). After a 4-h incubation, the Calcein AM release of supernatant from each well was measured. Fluorescence intensity was determined using a microplate reader (Power Scan HT) (BioTek Instruments) with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Cytolytic activity (as % of lysis) was calculated as: % lysis=(E-S)/(M-S) x100, where E is fluorescence of combined target and effector cells, S is spontaneous fluorescence of target cells only, and M is maximum fluorescence measured after lysing all cells with a buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), and 10 mM of EDTA.

Cells in DMEM supplemented with 10% FBS (2x10⁴ cells/well) were plated in 96-well plates., and incubated for 5 h at 37°C with either anti-HER2 antibodies or control IgG (mouse IgG_2b) (Sigma-Aldrich Corp.) and 10% of rabbit complement (Low-Tox-M Rabbit Complement) (Cedarlane Laboratories). To assess cell viability, an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; inner salt] assay was performed using a CellTiter 96 AQueous assay kit (Promega).

Sixteen six-week-old female BALB/c nude mice were purchased from Charles River (Kanagawa, Japan) and used at 10 weeks of age. BT-474 cells (0.3 ml of 1.33x10⁸ cells/ml in DMEM) were mixed with 0.5 ml BD Matrigel Matrix Growth Factor Reduced (BD Biosciences). One hundred-µl of this suspension (5x10⁶ cells) was injected subcutaneously into the left flank. After day 1, 100 µg H₂Mab-19 and control mouse IgG (Sigma-Aldrich Corp.) in 100 µl PBS were injected i.p. into treated and control mice, respectively. Additional antibodies were then injected on days 7 and 14. Eighteen days after cell implantation, all mice were euthanized by cervical dislocation and tumor diameters and volumes were determined as previously described (15).

Thirty-two six-week-old female BALB/c nude mice were purchased from Charles River and used at 10 weeks of age. HSC-2 or SAS cells in DMEM (0.3 ml with 1.33x10⁸ cells/ml) were mixed with 0.5 ml BD Matrigel Matrix Growth Factor Reduced (BD Biosciences). A 100-µl suspension containing 5x10⁶ cells was injected subcutaneously into the left flank. After day 1, 100 µg H₂Mab-19 and control mouse IgG (Sigma-Aldrich Corp.) in 100 µl PBS were injected i.p. into treated and control mice, respectively. Additional antibodies were then injected on days 6 and 14. Twenty days after cell implantation, all mice were euthanized by cervical dislocation. Tumor diameters and volumes were determined as previously described (15).

All data were expressed as mean ± SEM. Statistical analysis used ANOVA and Tukey-Kramer's test with GraphPad Prism 6 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

One mouse was immunized with the recombinant extracellular domain of HER2(16), purified using the MAP tag system (17). Flow cytometry was performed to check reactions with the LN229 cells (glioblastoma) and HER2-overexpressing LN229 cells (LN229/HER2). LN229 cells endogenously express HER2 and some reaction with these cells was expected. The overexpression of HER2 in LN229/HERS2 cells would produce a stronger reaction. One IgG_2b subclass clone of H₂Mab-19 was obtained, though almost all mAbs were in the mouse IgG₁ subclass. H₂Mab-19 reacted with LN229/HER2 and weakly reacted with LN229 cells (Fig. 1A), indicating that H₂Mab-19 is specific to HER2.

H₂Mab-19 recognized endogenous HER2 in a breast cancer cell line, BT-474, which is HER2-positive (18), but did not react with a breast cancer cell line, MDA-MB-468, which is HER2-negative (18) (Fig. 1A). Further, H₂Mab-19 strongly reacted with endogenous HER2 in HO-1-u-1 cells (oral cancer) and only weakly reacted with other oral cancer cell lines, Ca9-22, HSC-2, and SAS (Fig. 1A). Using flow cytometry, binding affinities (K_D) of H₂Mab-19 to BT-474, HSC-2, and SAS cell lines were 2.3x10^-8, 9.5x10^-9 and 5.5x10^-9 M, respectively. These results indicate that H₂Mab-19 maintains high affinity across HER2-expressing cell lines. H₂Mab-19 did not stain FFPE-breast cancer tissues (Fig. S1). In contrast, H₂Mab-19 reacted with frozen breast cancer tissues although the sensitivity of H₂Mab-77 was better than that of H₂Mab-19 (Fig. S2).

This study examined whether H₂Mab-19 induced ADCC and CDC in HER2-expressing breast or OSCC cell lines. H₂Mab-19 was a mouse IgG_2b subclass antibody that could possess both ADCC and CDC. H₂Mab-19 exhibited high ADCC activity against BT-474, HSC-2, and SAS cells (Fig. 2A). High CDC activity was also observed in BT-474, HSC-2, and SAS cells (Fig. 2B), suggesting that H₂Mab-19 might exert antitumor activity in vivo.

To study the antitumor activity of H₂Mab-19 on cell growth in vivo, BT-474 cells were implanted subcutaneously in the flanks of nude mice. H₂Mab-19 and control mouse IgG were injected i.p. three times (days 1, 7, and 14 after cell injection) into treated and control mice, respectively. Tumor formation was observed in mice in both H₂Mab-19-treated and control groups. H₂Mab-19 treatment significantly reduced tumor development compared to development in control mice on days 5, 7, 12, 15, and 18 (Fig. 3A, upper). Weights of tumors from H₂Mab-19-treated mice were significantly less than for tumors from IgG-treated control mice (Fig. 3B, upper). BT-474 xenografts on day 18 are shown in Fig. S3A. Resected tumors are depicted in Fig. S3B. Total body weight was not significantly different between the two groups (Fig. S3C). We could not show the histological data about the liver and kidney in this study. HER2 was highly expressed in all cancer cells of H₂Mab-19-treated BT-474 and control xenografts (Fig. S4).

H₂Mab-19 possessed antitumor activity in mouse xenografts of breast cancers. Whether this activity extended to xenografts of oral cancers was also assessed. BT-474 cells expressed high levels of HER2 (Fig. 1A); HER2 levels were however low in HSC-2 and SAS cells (Fig. 1B). Nevertheless, HSC-2 and SAS are useful for investigation of antitumor activity in vivo (16). Thus, HSC-2 and SAS were used for mouse xenografts of oral cancers.

Initially, HSC-2 cells were implanted subcutaneously into the flanks of nude mice. H₂Mab-19 and mouse IgG were injected i.p. three times (on days 1, 6, and 14 after cell injections into treated and control mice, respectively. Tumor formation was observed in mice in both groups. In comparison to control mice, H₂Mab-19-treated mice showed significantly reduced tumor development on days 6, 10, 14, 17 and 20 (Fig. 3A, middle). Weights of tumors from H₂Mab-19-treated mice were significantly less than for tumors from control mice (Fig. 3B, middle). HSC-2 xenograft mice are shown on day 20 in Fig. S5A and resected tumors are depicted in Fig. S5B. Total body weights were not significantly different between the two groups (Fig. S5C). We could not show the histological data about the liver and kidney in this study. HER2 was not expressed in cancer cells of H₂Mab-19-treated or control groups (Fig. S6). HER2 expression was diminished in HSC-2 xenografts, and H₂Mab-19 did not exert effective antitumor activity.

For the second xenograft model of oral cancers, SAS cells were subcutaneously implanted into the flanks of nude mice. H₂Mab-19 and mouse IgG were injected i.p. thrice, on days 1, 6, and 14 after cell injections into the mice, into treated and control mice, respectively. Tumor formation was observed in mice in both treated and control groups. In comparison to IgG-treated control mice, H₂Mab-19 significantly reduced tumor development on days 14, 17, and 20 (Fig. 3A, lower). Weights of tumors from H₂Mab-19-treated mice were significantly less than tumors from IgG-treated control Mice (Fig. 3B, lower). The SAS xenografts on day 20 are shown in Fig. S7A and resected tumors are depicted in Fig. S7B. Total body weights were not significantly different between the two groups (Fig. S7C). We could not show the histological data about the liver and kidney in this study. HER2 was not expressed in cancer cells of H₂Mab-19-treated and control groups (Fig. S8). HER2 expression was diminished in SAS xenografts, and H₂Mab-19 did not exert effective antitumor activity.