
Anti‑EGFR monoclonal antibody 134‑mG2a exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma
- Authors:
- Published online on: August 10, 2020 https://doi.org/10.3892/ijmm.2020.4700
- Pages: 1443-1452
-
Copyright: © Hosono et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
More than 350,000 individuals are diagnosed with oral cancer annually, and oral cancer will ultimately prove fatal in almost half of those diagnosed with the disease (1). Of the defined histological types of oral cancer, >90% of patients are diag-nosed with oral squamous cell carcinoma (OSCC), which typically arises on the lips or within the oral cavity (2). The most effective treatments currently available for OSCC depend on its clinical stage at presentation. Although stage-I and -II OSCCs are treated with surgery or radiotherapy, advanced stage-III and -IV disease is treated with a combination of surgery, radiotherapy and chemotherapy (3). Chemotherapeutic regimens typically include cisplatin as a first-line agent; it is often combined with docetaxel or 5-fluorouracil (4,5). Paclitaxel, methotrexate and carboplatin can be also used in the treatment of OSCCs (6); however, there is only limited information available on the efficacy of molecular targeting drugs and/or antibody-based therapies for OSCC.
The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases, and is involved in cell growth and differentiation (7-9). EGFR forms homo- or heterodimers with other HER family members, such as HER2 and HER3, and thereby activate downstream signaling cascades. These pathways are frequently dysregulated in malignant diseases, including OSCC, often via the overexpression of EGFR (10). Nimotuzumab is a humanized monoclonal antibody (mAb) directed against the extracellular domain of the EGFR that has been shown to have clinical efficacy in various types of cancer (11). Although nimotuzumab has been approved in 29 countries for use in the treatment of advanced head and neck carcinoma, esophageal cancer, nasopharyngeal carcinoma and pancreatic cancer, only modest success has been achieved with respect to the treatment of recurrent and/or metastatic OSCC (12). Although a number of EGFR-targeted therapies have been used in patients with OSCC, treatment failures due to the low response rates and acquired resistance have been reported (13).
In a previous study by the authors, mice were immunized with purified recombinant EGFR, and successfully produced monoclonal EMab-134 (mouse IgG1, kappa). This antibody detected endogenous EGFR in oral cancers in applications including flow cytometry, western blot analysis and immunohistochemistry (14). For example, when used in immunohistochemical analysis, EMab-134 reacted with its target antigen in 36 of 38 (94.7%) oral cancer specimens. The minimum epitope of EMab-134 was determined to be 377-RGDSFTHTPP-386 (15). Although EMab-134 has proven to be very useful for the detection of EGFR, the mouse IgG1 subclass does not facilitate antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activities.
To address this issue, in the present study, EMab-134 (IgG1 subclass) was converted into 134-mG2a of the mouse IgG2a subclass. It was then determined whether 134-mG2a exhibits ADCC, CDC, and in vivo antitumor activities against OSCCs.
Materials and methods
Antibodies
Anti-EGFR mAb EMab-134 (mouse IgG1, kappa) was developed as previously described (14). To generate 134-mG2a, VH cDNA of EMab-134 and CH mouse IgG2a were subcloned into pCAG-Ble vector, and VL and CL cDNAs of EMab-134 were subcloned into pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation), respectively. Vectors were transfected into ExpiCHO-S cells using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.). The resulting mAb, 134-mG2a, was purified with Protein G-Sepharose (GE Healthcare Bio-Sciences). Mouse IgG (cat. no. I8765), IgG1 (cat. no. M7894), and IgG2a (cat. no. M7769) were purchased from Sigma-Aldrich; Merck KGaA.
Cell lines
The CHO-K1 cell line was obtained from the American Type Culture Collection (ATCC). Human EGFR-expressing CHO-K1 cells (CHO/EGFR) were previously established by the transfection of pCAG/PA-EGFR-RAP-MAP into CHO-K1 cells using Lipofectamine LTX (Thermo Fisher Scientific, Inc.) (16). The amino acid sequences of each tag were as follows: PA tag (17), 12 amino acids (GVAMPGAEDDVV); RAP tag (18), 12 amino acids (DMVNPGLEDRIE); and MAP tag (19), 12 amino acids (GDGMVPPGIEDK). OSCC cell lines, including HSC-2 (oral cavity) and SAS (tongue) were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB). CHO-K1 and CHO/EGFR were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc.). The HSC-2 and SAS cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.). Cell culture medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 units/ml of penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Nacalai Tesque, Inc.). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
Animals
All animal experiments were performed in accordance with relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal experiments for ADCC and antitumor activity were approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (Permit. no. 2019-049 for ADCC assays, 2019-046 for antitumor experiments). Mice were maintained in a pathogen-free environment (23±2°C, 55±5% humidity) on an 11-h light/13-h dark cycle with food and water supplied ad libitum across the experimental period. Mice were monitored for health and weight every 2 or 5 days during the 3-week period of each experiment. The loss of original body weight to a point >25% and/or a maximum tumor size >3,000 mm3 were identified as humane endpoints for euthanasia. Mice were euthanized by cervical dislocation; death was verified by respiratory and cardiac arrest.
Flow cytometry
Cells were harvested by brief exposure to 0.25% trypsin/1 mM ethylenediamine tetra acetic 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 of anti-EGFR mAbs for 30 min at 4°C followed by Alexa Fluor 488-conjugated anti-mouse IgG at a dilution of 1:1,000 (cat. no. 4408S; Cell Signaling Technology, Inc.) for 30 min at 4°C. Fluorescence data were collected using an SA3800 Cell Analyzer (Sony Corp.).
Western blot analyses
Cell pellets were suspended using lysis buffer (1% Triton X-100 and 50 µg/ml aprotinin in PBS) on ice for 15 min. Following centrifugation (20,630 × g, 15 min, 4°C), cell lysates were boiled in sodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc.). The samples were electrophoresed on 5-20% polyacrylamide gels (Nacalai Tesque, Inc.) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA). After blocking with 4% skim milk (Nacalai Tesque, Inc.) for 1 h, the membranes were incubated with anti-EGFR mAbs or anti-β-actin (1 µg/ml) for 1 h, followed by incubation with HRP-conjugated anti-mouse immunoglobulins at a 1:2,000 dilution (Agilent Technologies, Inc.) for 1 h. The membranes were developed with the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager (DRC Co., Ltd.). All western blot analysis procedures were performed at room temperature.
Immunohistochemical analyses
Histological sections (4-µm-thick) of an oral cancer tissue array (cat. no. OR601c; US Biomax, Inc.) were directly autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. The sections were then incubated with 5 µg/ml anti-EGFR mAbs for 1 h at room temperature and treated using an Envision+ kit (Agilent Technologies, Inc.) for 30 min. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2 min, and the sections were then counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation). Hematoxylin and eosin (H&E) staining was performed using consecutive tissue sections as follows: Hematoxylin staining (FUJIFILM Wako Pure Chemical Corporation) for 5 min and eosin staining (FUJIFILM Wako Pure Chemical Corporation) for 2 min at room temperature. Leica DMD108 (Leica Microsystems GmbH) was used to examine the sections and obtain images.
Determination of the binding affinity
The cells were suspended in 100 µl of serially diluted anti-EGFR mAbs (0.6-10 µg/ml) followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000; Cell Signaling Technology, Inc.). Fluorescence data were collected using an EC800 Cell Analyzer (Sony Corp.). The dissociation constant (KD) was calculated by fitting binding isotherms to built-in one-site binding models in GraphPad Prism 7 (GraphPad Software, Inc.).
ADCC
A total of 6 female 6-week-old BALB/c nude mice (weighing 15-18 g) were purchased from Charles River Laboratories, Inc. Spleen cells from 6 mice were used as the source of natural killer (NK) cells for the evaluation of ADCC, which has been reported previously (20). Following euthanasia by cervical dislocation, the spleens were removed aseptically and single-cell suspensions were obtained by forcing spleen tissues through a sterile cell strainer (352360, BD Falcon, Corning, Inc.) 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; this preparation was used as effector cells. Target tumor cells were labeled with 10 µg/ml Calcein AM (Thermo Fisher Scientific, Inc.) and resuspended in the same medium. The target cells (2×104 cells/well) were plated in 96-well plates and mixed with effector cells (effector/target cell ratio, 50), 100 µg/ml of anti-EGFR antibodies or control IgGs. Following a 4-h incubation at 37°C, the release of Calcein AM into the supernatant was measured in each well. The fluorescence intensity was determined using a microplate reader (Power Scan HT; BioTek Instruments, Inc.) with an excitation wave-length of 485 nm and an emission wavelength of 538 nm. Cytolytic activity (% lysis) was calculated using the equation % lysis=(E-S)/(M-S) ×100, where 'E' is the fluorescence measured in combined cultures of target and effector cells, 'S' is the spontaneous fluorescence of target cells only, and 'M' is the maximum fluorescence measured following the lysis of all cells with a buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4) and 10 mM of EDTA.
CDC
The cells (2×104 cells/well) were plated in 96-well plates and mixed with rabbit complement (final dilution 1:10; Low-Tox-M Rabbit Complement; Cedarlane Laboratories) together with 100 µg/ml of anti-EGFR or control IgGs. Following 5 h incubation at 37°C, MTS [3-(4,5-dimethylthi-azol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; inner salt] assay was performed using a CellTiter 96 AQueous assay kit (Promega Corp.).
Antitumor activity of 134-mG2a in xenografts of CHO/EGFR cells
A total of 24 female BALB/c nude mice (5 weeks old, weighing 14-17 g) were purchased from Charles River Laboratories, Inc. and used in experiments once they reached 7 weeks of age. CHO/EGFR cells (0.3 ml of 1.33×108 cells/ml in DMEM) were mixed with 0.5 ml BD Matrigel Matrix Growth Factor Reduced (BD Biosciences); 100 µl of this suspension (5×106 cells) was injected subcutaneously into the left flanks of the mice. On day 1 post-inoculation, 100 µg of EMab-134 (n=8), 134-mG2a (n=8), or control mouse IgG (n=8) in 100 µl PBS were injected intraperitoneally. Additional antibody inoculations were performed on days 7 and 14. At 21 days following cell implantation, all mice were euthanized by cervical dislocation; tumor diameters and volumes were determined as previously described (21).
Antitumor activity of 134-mG2a in xenografts of oral cancers
A total of 48 female BALB/c nude mice (5 weeks old, weighing 14-17 g) were purchased from Charles River Laboratories, Inc. and used in experiments once they reached 7 weeks of age. The HSC-2 and SAS cells (0.3 ml of 1.33×108 cells/ml in DMEM) were mixed with 0.5 ml BD Matrigel Matrix Growth Factor Reduced (BD Biosciences); 100 µl of this suspension (5×106 cells) was injected subcutaneously into the left flanks of the mice. On day 1 post-inoculation, 100 µg of EMab-134 (n=8 in each group), 134-mG2a (n=8 in each group), or control mouse IgG (n=8 in each group) in 100 µl PBS were injected intraperitoneally. Additional antibody inoculations were performed on days 7 and 14. At 18 days following cell implantation, all mice were euthanized by cervical dislocation, and tumor diameters and volumes were determined.
Statistical analyses
All data are expressed as the means ± standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer's test using R statistical (R Foundation for Statistical Computing). A value of P<0.05 was adopted as a level of statistical significance.
Results
Generation and characterization of 134-mG2a, a mouse IgG2a-type anti-EGFR antibody
As mouse IgG2a subclass facilitates both ADCC and CDC (22), in the present study, a mouse IgG2a version of the IgG1 EMab-134 (14) was generated by subcloning VH cDNA of EMab-134 and CH mouse IgG2a into pCAG-Ble vector, and VL and CL cDNAs of EMab-134 into pCAG-Neo vector. The IgG2a version of EMab-134 was named 134-mG2a. The sensitivity of 134-mG2a in CHO/EGFR, HSC-2 and SAS cells was analyzed by flow cytometry. As shown in Fig. 1, EMab-134 and 134-mG2a were equally effective at detecting CHO/EGFR, HSC-2 and SAS cells using this method.
Subsequently, the sensitivities of EMab-134 and 134-mG2a were compared when used to probe lysates of CHO/EGFR, HSC-2 and SAS cells by western blot analysis. Notably, 134-mG2a exhibited a relatively higher reactivity when detecting their targets in cell lysates from CHO/EGFR and HSC-2 cells; both antibodies were faintly reactive against targets in SAS cell lysates (Fig. 2). The molecular weight of the EGFRs of HSC-2 and SAS cells was smaller than that expressed in CHO-K1 cells, as PA-EGFR-RAP-MAP, in which 3 peptide tags, such as PA tag, RAP tag, and MAP tag were added, was transfected into the CHO-K1 cells (16).
Immunohistochemical analysis revealed that both EMab-134 and 134-mG2a detected membrane antigens in oral cancer tissues (Fig. 3). 134-mG2a exhibited higher staining intensities when compared to the results from EMab-134 in several oral cancer tissues with various levels of EGFR expression; intensities included 1+ (Fig. 3A), 2+ (Fig. 3B) and 3+ (Fig. 3C). No staining was observed in tissues incubated with the buffer control.
A kinetic analysis of the interactions of EMab-134 and 134-mG2a with CHO/EGFR cells was then performed using flow cytometry. As shown in Fig. 4, the dissociation constant (KD) for the interaction of EMab-134 with CHO/EGFR cells was 3.2×10−9 M. By contrast, the KD for the interaction of 134-mG2a with CHO/EGFR cells was 2.1×10−9 M (Fig. 4). The binding affinity of 134-mG2a for CHO/EGFR cells was 1.5-fold higher than that of EMab-134; taken together with the results from western blot analysis, this result suggests that the higher binding affinity of 134-mG2a may result in the higher sensitivity observed in western blot and immunohistochemical analyses.
134-mG2a-mediated ADCC in oral cancer cell lines
Subsequently, whether the newly-developed 134-mG2a was capable of mediating ADCC against CHO/EGFR cells or oral cancer cell lines, including HSC-2 and SAS cells was examined. As shown in Fig. 5A, 134-mG2a elicited ADCC (66% cytotoxicity; P<0.01) against CHO/EGFR cells more effectively than did control mouse IgG2a (23% cytotoxicity). By contrast, EMab-134 promoted no significant ADCC (23% cytotoxicity; n.s.) against CHO/EGFR cells compared to that observed in response to control mouse IgG1 (23% cytotoxicity). Similarly, 134-mG2a elicited ADCC (53% cytotoxicity; P<0.01) against the HSC-2 cells more effectively than did control mouse IgG2a (13% cytotoxicity) (Fig. 5B). By contrast, EMab-134 elicited no significant ADCC (14% cytotoxicity; n.s.) against the HSC-2 cells compared to that observed in response to mouse IgG1 control (13% cytotoxicity). Furthermore, 134-mG2a elicited higher ADCC (63% cytotoxicity; P<0.01) against the SAS cells compared with that elicited by control mouse IgG2a (32% cytotoxicity; Fig. 5C), while EMab-134 elicited no significant ADCC (32% cytotoxicity; n.s.) against SAS cells compared to that observed in response to control mouse IgG1 (33% cytotoxicity). Taken together, the novel mAb 134-mG2a exhibited significantly higher ADCC for all 3 EGFR-expressing cell lines featured in the present study; by contrast, no ADCC was observed in response to EMab-134.
134-mG2a-mediated CDC in oral cancer cell lines
The present study then examined whether 134-mG2a induces CDC in CHO/EGFR cells or in oral cancer cell lines, including HSC-2 and SAS cells. As shown in Fig. 6A, 134-mG2a elicited a higher degree of CDC (46% cytotoxicity; P<0.01) in CHO/EGFR cells compared with that elicited by control mouse IgG2a (5.9% cytotoxicity). By contrast, EMab-134 elicited no significant CDC (11% cytotoxicity; n.s.) against CHO/EGFR cells compared to that observed in response to control mouse IgG1 (7.4% cytotoxicity). Similarly, 134-mG2a elicited a higher degree of CDC (79% cytotoxicity; P<0.01) against HSC-2 cells compared with that elicited by control mouse IgG2a (19% cytotoxicity; Fig. 6B). By contrast, EMab-134 elicited no significant CDC (20% cytotoxicity; n.s.) against the HSC-2 cells compared to that observed in response to control mouse IgG1 (19% cytotoxicity). Furthermore, 134-mG2a elicited a higher degree of CDC (60% cytotoxicity; P<0.01) against SAS cells compared with that elicited by control mouse IgG2a (15% cytotoxicity; Fig. 6C). By contrast, EMab-134 elicited no significant CDC (28% cytotoxicity; n.s.) against the SAS cells compared to that observed in response to control mouse IgG1 (20% cytotoxicity). Taken together, these results demonstrated that 134-mG2a promoted significantly higher levels of CDC against all EGFR-expressing cells evaluated in this study; by contrast, EMab-134 was not effective in this role. As the ADCC/CDC activities of 134-mG2a in oral cancer cells were all potent and effective, this antibody may also exert antitumor activity against oral cancer cells in vivo.
Antitumor activities of 134-mG2a in the mouse xenografts of CHO/EGFR cells
In the CHO/EGFR xenograft models, 134-mG2a (100 µg), EMab-134 (100 µg) and control mouse IgG (100 µg) were injected intraperitoneally into the mice on days 1, 7 and 14 following the injection of CHO/EGFR cells. The tumor volume was measured on days 7, 9, 14, 18 and 21 after the injection. The administration of 134-mG2a resulted in a significant reduction in tumor development on days 7 (P<0.01), 9 (P<0.01), 14 (P<0.05), 18 (P<0.01) and 21 (P<0.01) compared to the mice treated with either EMab-134 or control mouse IgG (Fig. 7). No significant differences in tumor volume were observed in a comparison between the EMab-134- and control IgG-treated mice on days 7, 9, 14, 18, and 21. The administration of 134-mG2a resulted in a 41% reduction in tumor volume compared to the EMab-134-treated mice on day 21 post-injection. Furthermore, tumors from the 134-mG2a-treated mice weighed significantly less than tumors from the EMab-134-treated mice (50% reduction; P<0.01, Fig. 8A). No significant differences in tumor weights were observed when comparing those from the EMab-134- and control mouse IgG-treated mice (Fig. 8A). Tumors that were resected from mice on day 21 are illustrated in Fig. 8B. Total body weights did not differ significantly among the 3 groups (data not shown). Taken together, these results indicated that the administration of 134-mG2a effectively reduced the growth of CHO/EGFR xenografts.
Antitumor activities of 134-mG2a in mouse xenografts of HSC-2 oral cancer cells
In the HSC-2 xenograft models, 134-mG2a (100 µg), EMab-134 (100 µg), or control mouse IgG (100 µg) were injected intraperitoneally into mice on days 1, 7 and 14 after the HSC-2 cell injections. Tumor volume was measured on days 7, 9, 14 and 18. The administration of 134-mG2a resulted in significantly decreased tumor development on days 7 (P<0.01), 9 (P<0.01), 14 (P<0.01) and 18 (P<0.01) in comparison to the EMab-134-treated mice (Fig. 9). No significant differences were observed between the EMab-134- and control IgG-treated mice on days 7, 9, 14 and 18. The administration of 134-mG2a resulted in a 57% reduction in tumor volume compared to the EMab-134-treated mice on day 18 post-injection. Tumors from the 134-mG2a-treated mice weighed significantly less than the tumors from the EMab-134-treated mice (37% reduction; P<0.01, Fig. 10A). No significant differences in tumor weight were observed when comparing those from the EMab-134- and control mouse IgG-treated mice. Tumors resected on day 18 are illustrated in Fig. 10B. Total body weights did not differ significantly among the 3 groups (data not shown). These results indicated that the administration of 134-mG2a effectively limited the growth of HSC-2 cell xenografts.
Antitumor activities of 134-mG2a in mouse xenografts of SAS oral cancer cells
In the SAS xenograft models, 134-mG2a (100 µg), EMab-134 (100 µg), and control mouse IgG (100 µg) were injected intraperitoneally into the mice on days 1, 7 and 14 after SAS cell injections. Tumor volumes were measured on days 7, 9, 14 and 18. The administration of 134-mG2a resulted in significantly reduced tumor development as determined on days 7 (P<0.01), 9 (P<0.01), 14 (P<0.01) and 18 (P<0.01) when compared to tumors from the EMab-134-treated mice (Fig. 11). No significant differences between EMab-134 and control mouse IgG-treated mice on days 7, 9, 14 and 18 were observed. The administration of 134-mG2a resulted in a 70% reduction in tumor volume on day 18 compared to the responses observed among the EMab-134-treated mice. Tumors from the 134-mG2a-treated mice weighed significantly less than the tumors from the EMab-134-treated mice (60% reduction; P<0.01, Fig. 12A). No significant differences in tumor weight were observed when comparing the tumors from the EMab-134- and control mouse IgG-treated mouse resected on day 18. The tumors resected from the mice are shown in Fig. 12B. Total body weights did not significantly differ among the three groups (data not shown). These results indicated that the administration of 134-mG2a effectively limited the growth of SAS xenografts.
Discussion
The authors of the present study previously established a sensitive and specific anti-EGFR mAb, EMab-134 (mouse IgG1), which is very useful for several applications, including flow cytometry, western blot analysis and immunohistochemistry (14). This antibody could not be used to investigate antitumor activity as the IgG1 subclass does not exhibit ADCC/CDC activities. Therefore, EMab-134 was converted into 134-mG2a (IgG2a subclass). It was demonstrated that 134-mG2a elicits both ADCC and CDC in vitro (Figs. 5 and 6), and antitumor activities against both CHO/EGFR xenografts (Figs. 7 and 8) and OSCC xenografts (Figs. 9-12) in vivo. Importantly, the administration of 134-mG2a efficiently reduced the growth of OSCC xenografts at all time points examined when compared to the responses to EMab-134. Nevertheless, only limited reductions in HSC-2 and SAS tumor volume were observed in response to the administration of 134-mG2a, to 57 and 70%, respectively. These results suggest that targeting EGFR with this antibody may not be sufficient to eliminate most OSCCs. The authors have previously added EGF to HSC-2 and SAS cell lines; however, these cell lines did not respond to EGF stimulation and did not grow well compared to the control cells (21). The results indicated that 134-mG2a and EMab-134 antibodies could not neutralize the EGF-EGFR axis. Taken together, antitumor activities by 134-mG2a were exerted by ADCC and CDC activities, not neutralization.
In a previous study, it was found that HER2 was expressed in oral cancers, and that the administration of an anti-HER2 mAb (clone H2Mab-19, mouse IgG2b) resulted in antitumor activity against HSC-2 and SAS xenografts (23). By contrast, Mirza et al (24) demonstrated that only one case out of 140 OSCCs was HER2-positive; as such, the feasibility of anti-HER2 therapy for OSCC remains uncertain. In another study, the authors developed a sensitive and specific mAb against EGFR that recognized a distinct epitope (clone EMab-17, mouse IgG2a) and that elicited both ADCC and CDC, as well as antitumor activity against HSC-2 and SAS xenografts (21). The extent of ADCC, CDC or antitumor activities of EMab-17 and 134-mG2a were not yet compared, nor was the binding epitope of EMab-17 determined; further investigations are warranted in order to select the optimal anti-EGFR mAb for the treatment of OSCCs.
The authors previously converted an anti-podocalyxin (PODXL) mAb of IgG1 subclass (PcMab-47) into a mouse IgG2a-type mAb (47-mG2a) to facilitate the evaluation of ADCC and CDC (25). The authors also developed 47-mG2a-f, a core fucose-deficient variant of 47-mG2a in order to increase its ADCC. In vivo analysis revealed that 47-mG2a-f, but not 47-mG2a, exerted antitumor activity in HSC-2 and SAS xenograft models at administered 3 times at doses of 100 µg/mouse/week; these results indicated that a core fucose-deficient anti-PODXL mAb may also be useful for antibody-based therapy against PODXL-expressing OSCCs. Moreover, a cancer-specific mAb (CasMab) against podoplanin (PDPN) was established, which is expressed in a number of types of cancer, including oral cancers (26). In xenograft models of HSC-2 cells, a mouse-human chimeric mAb, chLpMab-23, exerted antitumor activity by engaging human NK cells; these results suggest that chLpMab-23 may be advantageous for antibody therapy against PDPN-expressing oral cancers (27). Antibody regimens that focus on multiple targets, including EGFR, HER2, PODXL and PDPN, may ultimately be effective with the goal of conquering oral cancers. In the future, cancer-specific anti-EGFR mAbs may also be developed that can reduce the adverse effects of traditional antibody therapy.
Acknowledgments
The authors would like to thank Ms. Saori Handa, Ms. Saki Okamoto and Mr. Yu Komatsu (Department of Antibody Drug Development, Tohoku University Graduate School of Medicine) for providing technical assistance with the in vitro experiments, and Ms. Akiko Harakawa (Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation) for providing technical assistance with the animal experiments.
Abbreviations:
ADCC |
antibody-dependent cellular cytotoxicity |
ATCC |
American Type Culture Collection |
CasMab |
cancer-specific monoclonal antibody |
CDC |
complement-dependent cytotoxicity |
CHO |
Chinese hamster ovary |
DAB |
3,3′-diaminobenzidine tetrahydrochloride |
DMEM |
Dulbecco's modified Eagle's medium |
EDTA |
ethylenediaminetetraacetic acid |
EGFR |
epidermal growth factor receptor |
FBS |
fetal bovine serum |
JCRB |
Japanese Collection of Research Bioresources Cell Bank |
HER |
human epidermal growth factor receptor |
mAb |
monoclonal antibody |
n.s. |
not significant |
OSCC |
oral squamous cell carcinoma |
PBS |
phosphate-buffered saline |
PDPN |
podoplanin |
PODXL |
podocalyxin |
RPMI |
Roswell Park Memorial Institute |
PVDF |
polyvinylidene difluoride |
SEM |
standard error of the mean |
Funding
The present study was supported in part by the Japan Agency for Medical Research and Development (AMED) under the grant nos. JP20am0401013 (to YK), JP20am0101078 (to YK) and JP20ae0101028 (to YK), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant no. 17K07299 (to MKK), grant no. 19K07705 (to YK), and grant no. 20K16322 (to MS).
Availability of data and materials
The datasets used and/or analyzed during the study are available from the corresponding author on reasonable request.
Authors' contributions
HHo, TO, JT, TN, MS TA, YS and MY performed the experiments. MKK analyzed the experimental data. MK, HHa and YK conceived and designed the present study and wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Animal experiments for ADCC and the antitumor activity were approved by the institutional committee for experiments of the Institute of Microbial Chemistry (Permit. no. 2019-049 for ADCC assays, 2019-046 for antitumor experiments). The tissues used were from a purchased tissue microarray.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA and Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 68:394–424. 2018. View Article : Google Scholar : PubMed/NCBI | |
Rivera C: Essentials of oral cancer. Int J Clin Exp Pathol. 8:11884–11894. 2015.PubMed/NCBI | |
Guneri P and Epstein JB: Late stage diagnosis of oral cancer: Components and possible solutions. Oral Oncol. 50:1131–1136. 2014. View Article : Google Scholar : PubMed/NCBI | |
Vokes EE: Induction chemotherapy for head and neck cancer: Recent data. Oncologist. 15:3–7. 2010. View Article : Google Scholar : PubMed/NCBI | |
Marcazzan S, Varoni EM, Blanco E, Lodi G and Ferrari M: Nanomedicine, an emerging therapeutic strategy for oral cancer therapy. Oral Oncol. 76:1–7. 2018. View Article : Google Scholar : PubMed/NCBI | |
Furness S, Glenny AM, Worthington HV, Pavitt S, Oliver R, Clarkson JE, Macluskey M, Chan KK and Conway DI: Interventions for the treatment of oral cavity and oropharyngeal cancer: Chemotherapy. Cochrane Database Syst Rev. 13:CD0063862011. | |
Dokala A and Thakur SS: Extracellular region of epidermal growth factor receptor: A potential target for anti-EGFR drug discovery. Oncogene. 36:2337–2344. 2017. View Article : Google Scholar | |
Ogiso H, Ishitani R, Nureki O, Fukai S, Yamanaka M, Kim JH, Saito K, Sakamoto A, Inoue M, Shirouzu M and Yokoyama S: Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell. 110:775–787. 2002. View Article : Google Scholar : PubMed/NCBI | |
Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A, Schlessinger J and Waterfield MD: Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature. 307:521–527. 1984. View Article : Google Scholar : PubMed/NCBI | |
Mendelsohn J: The epidermal growth factor receptor as a target for cancer therapy. Endocr Relat Cancer. 8:3–9. 2001. View Article : Google Scholar : PubMed/NCBI | |
Schultheis B, Reuter D, Ebert MP, Siveke J, Kerkhoff A, Berdel WE, Hofheinz R, Behringer DM, Schmidt WE, Goker E, et al: Gemcitabine combined with the monoclonal antibody nimotuzumab is an active first-line regimen in KRAS wildtype patients with locally advanced or metastatic pancreatic cancer: A multicenter, randomized phase IIb study. Ann Oncol. 28:2429–2435. 2017. View Article : Google Scholar : PubMed/NCBI | |
Cohen RB: Current challenges and clinical investigations of epidermal growth factor receptor (EGFR)- and ErbB family-targeted agents in the treatment of head and neck squamous cell carcinoma (HNSCC). Cancer Treat Rev. 40:567–577. 2014. View Article : Google Scholar | |
Ma H, Jin S, Yang W, Zhou G, Zhao M, Fang S, Zhang Z and Hu J: Interferon-alpha enhances the antitumour activity of EGFR-targeted therapies by upregulating RIG-I in head and neck squamous cell carcinoma. Br J Cancer. 118:509–521. 2018. View Article : Google Scholar : PubMed/NCBI | |
Itai S, Yamada S, Kaneko MK, Chang YW, Harada H and Kato Y: Establishment of EMab-134, a sensitive and specific anti-epidermal growth factor receptor monoclonal antibody for detecting squamous cell carcinoma cells of the oral cavity. Monoclon Antib Immunodiagn Immunother. 36:272–281. 2017. View Article : Google Scholar : PubMed/NCBI | |
Kaneko MK, Yamada S, Itai S, Chang YW, Nakamura T, Yanaka M and Kato Y: Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134. Biochem Biophys Rep. 14:54–57. 2018.PubMed/NCBI | |
Itai S, Kaneko MK, Fujii Y, Yamada S, Nakamura T, Yanaka M, Saidoh N, Handa S, Chang YW, Suzuki H, et al: Development of EMab-51, a sensitive and specific anti-epidermal growth factor receptor monoclonal antibody in flow cytometry, western blot, and immunohistochemistry. Monoclon Antib Immunodiagn Immunother. 36:214–219. 2017. View Article : Google Scholar : PubMed/NCBI | |
Fujii Y, Kaneko M, Neyazaki M, Nogi T, Kato Y and Takagi J: PA tag: A versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin. Protein Expr Purif. 95:240–247. 2014. View Article : Google Scholar : PubMed/NCBI | |
Fujii Y, Kaneko MK, Ogasawara S, Yamada S, Yanaka M, Nakamura T, Saidoh N, Yoshida K, Honma R and Kato Y: Development of RAP tag, a novel tagging system for protein detection and purification. Monoclon Antib Immunodiagn Immunother. 36:68–71. 2017. View Article : Google Scholar : PubMed/NCBI | |
Fujii Y, Kaneko MK and Kato Y: MAP Tag: A novel tagging system for protein purification and detection. Monoclon Antib Immunodiagn Immunother. 35:293–299. 2016. View Article : Google Scholar : PubMed/NCBI | |
Kawada M, Inoue H, Kajikawa M, Sugiura M, Sakamoto S, Urano S, Karasawa C, Usami I, Futakuchi M and Masuda T: A novel monoclonal antibody targeting coxsackie virus and adenovirus receptor inhibits tumor growth in vivo. Sci Rep. 7:404002017. View Article : Google Scholar : PubMed/NCBI | |
Takei J, Kaneko MK, Ohishi T, Kawada M, Harada H and Kato Y: A novel anti-EGFR monoclonal antibody (EMab-17) exerts antitumor activity against oral squamous cell carcinomas via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Oncol Lett. 19:2809–2816. 2020.PubMed/NCBI | |
Kato Y, Kunita A, Fukayama M, Abe S, Nishioka Y, Uchida H, Tahara H, Yamada S, Yanaka M, Nakamura T, et al: Antiglycopeptide mouse monoclonal antibody LpMab-21 exerts antitumor activity against human podoplanin through anti-body-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Monoclon Antib Immunodiagn Immunother. 36:20–24. 2017. View Article : Google Scholar : PubMed/NCBI | |
Takei J, Kaneko MK, Ohishi T, Kawada M, Harada H and Kato Y: H2Mab-19, an anti-human epidermal growth factor receptor 2 monoclonal antibody exerts antitumor activity in mouse oral cancer xenografts. Exp Ther Med. 20:846–853. 2020. View Article : Google Scholar : PubMed/NCBI | |
Mirza S, Hadi N, Pervaiz S, Khan SZ, Mokeem SA, Abduljabbar T, Al-Hamoudi N and Vohra F: Expression of HER-2/neu in oral squamous cell carcinoma. Asian Pac J Cancer Prev. 21:1465–1470. 2020. View Article : Google Scholar : PubMed/NCBI | |
Itai S, Ohishi T, Kaneko MK, Yamada S, Abe S, Nakamura T, Yanaka M, Chang YW, Ohba SI and Nishioka Y: Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma. Oncotarget. 9:22480–22497. 2018. View Article : Google Scholar : PubMed/NCBI | |
Kato Y and Kaneko MK: A cancer-specific monoclonal antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep. 4:59242014. View Article : Google Scholar : PubMed/NCBI | |
Kaneko MK, Nakamura T, Kunita A, Fukayama M, Abe S, Nishioka Y, Yamada S, Yanaka M, Saidoh N, Yoshida K, et al: ChLpMab-23: Cancer-specific human-mouse chimeric anti-podoplanin antibody exhibits antitumor activity via antibody-dependent cellular cytotoxicity. Monoclon Antib Immunodiagn Immunother. 36:104–112. 2017. View Article : Google Scholar : PubMed/NCBI |