HSC-2 and SAS were obtained from the Japanese Collection of Research Bioresources Cell Bank. Chinese hamster ovaries (CHO)-K1, P3X63Ag8U.1 (P3U1), and LN229 were obtained from the American Type Culture Collection. LN229/EGFR (a stable transfectant) was previously produced by transfecting pCAG/PA-EGFR-RAP-MAP (14) into LN229 cells using the Neon Transfection System (Thermo Fisher Scientific, Inc.), and EGFR upregulation was demonstrated by Western blot analysis using anti-EGFR mAb, clone EMab-51 (15). P3U1 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.), while LN229, LN229/EGFR, 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 (FBS) (Thermo Fisher Scientific, Inc.), 100 units/ml of penicillin, 100 µg/ml of streptomycin, and 25 µg/ml of amphotericin B (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air.

All animal experiments were performed in accordance with relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal experiments described in the hybridoma production were approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2016MdA-153). Mice were monitored for health every day. Animal studies for the antitumor activity were approved by the institutional committee for experiments of the Institute of Microbial Chemistry (Permit number: 2019-014). Mice were monitored for health and weight every 3 or 4 days. The duration of the experiment was three weeks. A body weight loss exceeding 25% of total body weight and a maximum tumor size exceeding 3,000 mm³ were defined as a humane endpoint.

One four-week-old female BALB/c mouse was purchased from CLEA Japan and housed under specific pathogen-free conditions. Anti-EGFR hybridomas were produced, as previously mentioned (15). The ectodomain of EGFR with N-terminal PA tag (16), C-terminal RAP tag (17), and MAP tag (14) (EGFRec) was purified from the supernatant of LN229/EGFRec using the anti-RAP tag previously described (17).

One BALB/c mouse was immunized by intraperitoneal injections of LN229/EGFR together with Imject Alum (Thermo Fisher Scientific, Inc.). After several additional immunizations, a booster injection was intraperitoneally administered 2 days before harvesting spleen cells. Spleen cells were then fused with P3U1 cells using GenomONE-CF (Ishihara Sangyo Kaisha, Ltd.). The resulting hybridomas were cultured in an RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.). Culture supernatants were screened using enzyme-linked immunosorbent assays (ELISA) with a recombinant EGFR-extracellular domain. mAbs were purified from the supernatants of hybridomas, cultured in Hybridoma-SFM medium (Thermo Fisher Scientific, Inc.) using Protein G Sepharose 4 Fast Flow (GE Healthcare UK, Ltd.).

Recombinant proteins were immobilized on Nunc MaxiSorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at 1 µg/ml for 30 min. After blocking using a SuperBlock buffer (Thermo Fisher Scientific Inc.), the plates were incubated with primary antibodies, followed by 1:2,000 diluted peroxidase-conjugated anti-mouse IgG (Agilent Technologies). The enzymatic reaction was produced using a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc.).

Cells were harvested by brief exposure to 0.25% trypsin/1-mM ethylenediaminetetraacetic acid (EDTA) (Nacalai Tesque, Inc.). The cells were washed with 0.1% bovine serum albumin (BSA)/phosphate-buffered saline (PBS) and treated with 1 µg/ml of anti-EGFR mAbs for 30 min at 4°C, followed by Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000; Cell Signaling Technology, Inc.). Fluorescence data was collected using EC800 Cell Analyzers (Sony Corp.).

SAS (2×10⁵ cells) was suspended in 100 µl of serially diluted mAbs (6 ng/ml-100 µg/ml); Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000) (Cell Signaling Technology, Inc.) was then added, and fluorescence data was collected using a cell analyzer (EC800) (Sony Corp.). The dissociation constants (K_D) were computed by fitting the binding isotherms using the 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, and spleens were removed aseptically, and single-cell suspensions were obtained by dispersing the spleens using a syringe and pressing through stainless steel mesh. Erythrocytes were effectively lysed by 10-s exposure to ice-cold distilled water. Splenocytes were washed with DMEM and resuspended in DMEM with 10% FBS as effector cells. Target cells were labeled with 10-µg/ml Calcein AM (Thermo Fisher Scientific, Inc.) and resuspended in the medium. The target cells (2×10⁴ cells/well) were placed in 96-well plates and mixed with effector cells, anti-EGFR antibodies, or control IgG (mouse IgG_2a) (Sigma-Aldrich Corp.). After a 4-h incubation period, the Calcein AM release of supernatant from each well was measured. The fluorescence intensity was determined at an excitation wavelength of 485 nm and an emission wavelength of 538 nm using a microplate reader (Power Scan HT) (BioTek Instruments). Cytolytic activity (as % of lysis) was calculated using the following formula: % lysis=(E-S)/(M-S) ×100 (where E is the fluorescence released in the experimental cultures of target and effector cells, S is the spontaneous fluorescence released in cultures containing only target cells, and M is the maximum fluorescence obtained by adding a lysis buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), and 10 mM of EDTA to the target cells in order to lyse all cells).

HSC-2 and SAS cells were placed in 96-well plates of 2×10⁴ cells/well in DMEM supplemented with 10% FBS. Cells were incubated with either anti-EGFR antibodies or the control IgG (mouse IgG_2a) (Sigma-Aldrich Corp.) and 10% of rabbit complement (Low-Tox-M Rabbit Complement) (Cedarlane Laboratories) for 5 h at 37°C. To assess cell viability, MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxym-ethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; inner salt] assay was performed using a CellTiter 96 AQueous assay kit (Promega Corp.).

HSC-2 and SAS cells were washed with DMEM lacking FBS to eliminate the growth factors present in the enriched medium. Then, the cells were plated in 96-well culture plates at a density of 2,000 cells per well in 100 µl of 0.1% dialyzed FBS with or without 50 ng/ml of EGF (PeproTech). MTS assay was performed after 12, 24, and 36 h.

Thirty-two six-week-old female BALB/c nude mice were purchased from Charles River and used in experiments at 7 weeks of age. HSC-2 or SAS (0.3 ml of 1.33×10⁸/ml in RPMI) was mixed with 0.5 ml of BD Matrigel Matrix Growth Factor Reduced (BD Biosciences). A 100-µl suspension (containing 5×10⁶ cells) was injected subcutaneously into the left flanks of nude mice. After day 1, 100 µg of EMab-17 and control mouse IgG (Sigma-Aldrich Corp.) in 100 µl of PBS was injected into the peritoneal cavity of each mouse, followed by additional antibody injections on days 7 and 14. Mice were monitored for health and weight every 3 or 4 days. The diameter and volume of the tumor were determined as previously described (18), and the mice were euthanized 21 days after cell implantation. The duration of the experiment was three weeks. A body weight loss exceeding 25% of total body weight was defined as a humane endpoint. All data was expressed as mean ± SEM, and statistical analysis was conducted using Tukey-Kramer's test; P<0.05 was considered to indicate a statistically significant difference.

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

In this study, one mouse was immunized with LN229/EGFR, and culture supernatants of hybridoma were screened for binding to purified EGFRec using ELISA. Flow cytometry was used as a second screening to assess reactions with LN229 and LN229/EGFR cells. LN229 cells express endogenous EGFR (15); therefore, a stronger reaction against LN229/EGFR was required. One clone was obtained-EMab-17 of IgG_2a subclass-although almost all mAbs were determined to be a mouse IgG₁ subclass like EMab-51 (15) or EMab-134 (12).

Flow cytometry was used to demonstrate a stronger reaction with EMab-17 LN229/EGFR than with endogenous EGFR-expressing LN229 brain tumor cells (Fig. 1A), which indicated that EMab-17 is EGFR-specific. As a positive control, EMab-51 demonstrated a similar reaction with LN229 and LN229/EGFR (Fig. 1B). Endogenous HSC-2 and HSC-3 OSCC cell lines were also identified with both EMab-17 and EMab-51 (Fig. 1C and D). Flow cytometry was again applied to determine the binding affinity of EMab-17 for SAS cells (Fig. 2) and calculated K_D values for EMab-17 of 5.0×10⁻⁹ M against SAS. Similarly, K_D values were determined for EMab-51 as 6.3×10⁻⁹ M against SAS (Fig. 2), revealing that both EMab-17 and EMab-51 possess a high affinity for EGFR-expressing cell lines.

This study examined whether EMab-17 induced ADCC and CDC in EGFR-expressing OSCC cell lines. EMab-17 was determined to be a mouse IgG_2a subclass that might possess both ADCC and CDC (although mouse IgG₁ such as EMab-51 and EMab-134 does not) (12,15). As detailed in Fig. 3A, EMab-17 exhibited high ADCC activity against HSC-2 and SAS. Furthermore, high CDC activity was also observed for HSC-2 and SAS by EMab-17 (Fig. 3B), suggesting that EMab-17 might exert antitumor activities in vivo. Although we added EGF to SAS and HSC-2, these cell lines did not grow well compared to control cells by responding to EGF stimulation (data not shown), indicating that EMab-17 could not neutralize EGF-EGFR axis.

HSC-2 cells were subcutaneously implanted into the flanks of nude mice in order to study the antitumor activity of EMab-17 on cell growth in vivo. EMab-17 and the control mouse IgG were injected (on days 1, 7, and 14 after the cell injections) three times into the peritoneal cavity. Tumor formation was observed in mice from the control and EMab-17-treated groups in HSC-2 ×enograft models. EMab-17 demonstrated significant reduction in tumor development of the HSC-2 ×enograft compared with that in the control mouse IgG group on days 7, 10, 14, 17, and 21 (Fig. 4A). Mice treated with EMab-17 had significantly lower tumor weights compared to the control mouse IgG group in HSC-2 ×enograft models (Fig. 4B). The resected tumors of HSC-2 ×enografts are shown in Fig. 4C. The body weights of the HSC-2 ×enograft mice were recorded for 21 days (Fig. S1A). Body weight did not vary significantly between the two groups in the HSC-2 ×enograft models.

Identical experiments were performed using SAS xenograft models. EMab-17 demonstrated significantly reduced tumor development in the SAS xenograft group compared with that in the control mouse IgG group on days 7, 10, 14, 17, and 21 (Fig. 4D). The tumor weight of EMab-17-treated mice was significantly lower compared to the control mouse IgG group in the SAS xenograft models (Fig. 4E); the resected tumors of the SAS xenografts are illustrated in Fig. 4F. The body weights of the SAS xenograft mice were recorded for 21 days (Fig. S1B). The body weight did not vary significantly between the two SAS xenograft model groups.

Combined results confirm that EMab-17 exerted an antitumor activity against HSC-2 and SAS xenograft models via ADCC and CDC activities.