Contributed equally
CD44 is widely expressed on the surface of most tissues and all hematopoietic cells, and regulates many genes associated with cell adhesion, migration, proliferation, differentiation, and survival. CD44 has also been studied as a therapeutic target in several cancers. Previously, an anti-CD44 monoclonal antibody (mAb), C44Mab-5 (IgG1, kappa) was established by immunizing mice with CD44-overexpressing Chinese hamster ovary (CHO)-K1 cells. C44Mab-5 recognized all CD44 isoforms, and showed high sensitivity for flow cytometry and immunohistochemical analysis in oral cancers. However, as the IgG1 subclass of C44Mab-5 lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), the antitumor activity of C44Mab-5 could not be determined. In the present study, we converted the mouse IgG1 subclass antibody C44Mab-5 into an IgG2a subclass antibody, 5-mG2a, and further produced a defucosylated version, 5-mG2a-f, using FUT8-deficient ExpiCHO-S (BINDS-09) cells. Defucosylation of 5-mG2a-f was confirmed using fucose-binding lectins, such as AAL and PhoSL. The dissociation constants (
Oral cancers account for about 2% of all cancer cases diagnosed worldwide (
CD44 is known to be expressed in many cell types, including epithelial cells, fibroblasts, endothelial cells, and leukocytes (
One of the CD44 variants, CD44v6, was first identified as contributing to cancer metastasis, and CD44v6-specific monoclonal antibodies (mAbs) were found to inhibit metastasis of rat pancreatic cancers (
Many mAbs have been developed to target CD44 (
In this study, we converted the IgG1 subclass C44Mab-5 into a mouse IgG2a subclass mAb, 5-mG2a, and further produced a defucosylated version, 5-mG2a-f, using FUT8-deficient ExpiCHO-S cells (
Oral squamous carcinoma cell lines including HSC-2 (oral cavity) and SAS (tongue) were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB; Osaka, Japan). Chinese hamster ovary (CHO)-K1 was obtained from the American Type Culture Collection (ATCC). CD44v3-10 plus N-terminal PA16 tag-overexpressed CHO-K1 (CHO/PA16-CD44v3-10) was generated by transfection of pCAG/PA16-CD44v3-10 to CHO-K1 cells using the Neon Transfection System (Thermo Fisher Scientific, Inc.). The PA16 tag consists of 16 amino acids (GLEGGVAMPGAEDDVV) (
Mouse anti-CD44s mAb C44Mab-5 (IgG1, kappa) was developed as previously described (
All animal experiments were performed in accordance with relevant guidelines (e.g. ARRIVE guidelines) and regulations (e.g. 3R regulations) to minimize animal suffering and distress in the laboratory (
C44Mab-5 and 5-mG2a-f were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.) at 1 µg/ml for 30 min. After blocking using SuperBlock buffer (Thermo Fisher Scientific Inc.) containing 0.5 mM CaCl2, the plates were incubated with biotin-labeled lectins, such as
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 (BSA) in phosphate-buffered saline (PBS), cells were treated with primary mAbs 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 (4-µm thick) of an oral cancer tissue microarray (catalogue number: OR481; US Biomax Inc.) were directly autoclaved in citrate buffer (pH 6.0; Agilent Technologies Inc.) for 20 min. Sections were then incubated with 1 µg/ml primary mAbs for 1 h at room temperature and treated using an Envision+ Kit (Agilent Technologies) for 30 min. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min, and sections were then counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation). Hematoxylin and eosin (H&E) staining (FUJIFILM Wako Pure Chemical Corporation) was performed using consecutive tissue sections. Leica DMD108 (Leica Microsystems GmbH) was used to examine the sections and obtain images.
Cells were suspended in 100 µl of serially diluted mAbs (0.3 ng/ml-5 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 (
Cell lysates (10 µg) were boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). Proteins were separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA). After blocking with 4% skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% Tween 20, the membranes were incubated with 10 µg/ml of an anti-CD44 mAb [clone C44Mab-46 (mouse IgG1, kappa)]; available from Antibody Bank of Tohoku University (ABTU; Miyagi, Japan);
Total RNAs were prepared from cell lines using an RNeasy Mini Prep Kit (Qiagen Inc.). The initial cDNA strand was synthesized using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Inc.) by priming nine random oligomers and an oligo(dT) primer according to the manufacturer's instructions. We performed 35 cycles of PCR for amplification using HotStarTaq DNA Polymerase (Qiagen Inc.) with 0.2 µM of primer sets: Human CD44 sense (5′-GAAAGGAGCAGCACTTCAGG-3′), human CD44 antisense (5′-ACTGCAATGCAAACTGCAAGC-3′), GAPDH sense (5′-CAATGACCCCTTCATTGACC-3′), and GAPDH antisense (5′-GTCTTCTGGGTGGCAGTGAT-3′).
Six six-week-old female BALB/c nude mice were purchased from Charles River (Kanagawa, Japan). After euthanization by cervical dislocation, spleens were removed aseptically and single-cell suspensions 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 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 (2×104 cells/well) were plated in 96-well plates and mixed with effector cells, anti-CD44s antibodies, or control IgG (mouse IgG2a) (Sigma-Aldrich Corp.; Merck KGaA). After a 5-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 using the equation: % lysis=(E-S)/(M-S) ×100, where E is the fluorescence of the combined target and effector cells, S is the spontaneous fluorescence of the target cells only, and M is the 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 were plated in 96-well plates (2×104 cells/well), and incubated for 5 h at 37°C with either anti-CD44s antibodies or control IgG (mouse IgG2a) (Sigma-Aldrich Corp.; Merck KGaA) and 10% 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 Corp.).
3D cell proliferation was measured with the CellTiter-Glo® 3D cell viability assay (Promega Corp.) according to the manufacturer's instructions. Briefly, the cells were plated (2,000 cells/100 µl/well) in triplicate in 96-well ultra low attachment plates (Corning Inc.) with PBS or 100 µg/ml of mouse IgG2a and an anti-CD44 mAb (5-mG2a-f) in DMEM containing 10% FBS. The cell viability was measured after 48 h of incubation. The CellTiter-Glo® 3D reagent was added into wells in a 1:1 dilution (100 µl volume in well:100 µl of reagent) and then the plates were shaken for 5 min on an orbital shaker and incubated at room temperature for an additional 25 min. The luminescent signal was read using an EnSpire multi-plate reader (Perkin Elmer). Images were taken using an Evolution MP camera (Media Cybernetics). The proliferation rate was calculated relative to the control (PBS was added instead of the antibodies).
Sixty-four six-week-old female BALB/c nude mice were purchased from Charles River (Kanagawa, Japan) and used at 10 weeks of age. 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). One hundred microliters of this suspension (5×106 cells) was injected subcutaneously into the left flank. After day 1 (protocol-1) or day 7 (protocol-2), 100 µg of 5-mG2a-f and control mouse IgG (Sigma-Aldrich Corp.; Merck KGaA) in 100 µl PBS were injected intraperitoneally (i.p.) into treated and control mice, respectively. Additional antibodies were then injected on days 7 and 14 (protocol-1) or on days 14 and 21 (protocol-2). Nineteen days (protocol-1) or 27 days (protocol-2) after cell implantation, all mice were euthanized by cervical dislocation and tumor diameters and volumes were determined as previously described (
All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was carried out using ANOVA following Tukey-Kramer's test for ADCC and CDC. Sidak's multiple comparisons test was used for tumor volume and mouse weight, or Welch's t test for tumor weight and 3D cell proliferation assay using GraphPad Prism 7 (GraphPad Software, Inc.). P<0.05 was adopted as a level of statistical significance.
As mouse IgG2a possesses high ADCC and CDC activities (
We examined the sensitivity of 5-mG2a-f in CHO cells expressing CD44v3-10 plus N-terminal PA16 tag (CHO/PA16-CD44v3-10) and in oral squamous cell carcinoma (OSCC) cell lines (SAS and HSC-2) using flow cytometry. Both C44Mab-5 and 5-mG2a-f reacted with CHO/PA16-CD44v3-10 cells (
As shown in
Next, we performed immunohistochemical analysis on oral cancer cell lines. Representative images are shown in
We performed a kinetic analysis of the interactions of recC44Mab-5 and 5-mG2a-f with SAS and HSC-2 oral cancer cell lines using flow cytometry. As shown in
Because the mouse IgG1 subclass of C44Mab-5 does not possess ADCC or CDC activities, we synthesized a mouse IgG2a subclass mAb, and further defucosylated it to augment those activities. In this study, we examined whether the developed 5-mG2a-f induced ADCC and CDC in CD44-expressing oral cancer cell lines, such as SAS and HSC-2 cells. As shown in
Next, we investigated whether 5-mG2a-f inhibits cell growth of SAS and HSC-2 cells in anchorage-independent condition. As shown in
SAS cells were subcutaneously implanted into the flanks of nude mice. In protocol-1, 5-mG2a-f (100 µg) and control mouse IgG (100 µg) were injected i.p. three times into the mice, on days 1, 7, and 14 after SAS cell injections. Tumor volume was measured on days 6, 12, 15, and 19. Tumor development was significantly reduced in the 5-mG2a-f-treated mice on days 12, 15, and 19 in comparison to the IgG-treated control mice (
In protocol-2 of the SAS xenograft models, tumor formation of 16 SAS-bearing mice was observed on day 7. Then, these 16 SAS-bearing mice were divided into a 5-mG2a-f-treated group and a control group. On days 7, 14, and 21 after SAS cell injections into the mice, 5-mG2a-f (100 µg) and control mouse IgG (100 µg) were injected i.p. into the mice. Tumor formation was observed in mice in both treated and control groups. Tumor volume was measured on days 7, 12, 15, 19, 22, and 27. 5-mG2a-f-treated mice displayed significantly reduced tumor development on days 22 and 27 in comparison to IgG-treated control mice (
In a second xenograft model of oral cancers, HSC-2 cells were subcutaneously implanted into the flanks of nude mice. In protocol-1 of HSC-2 ×enograft models, 5-mG2a-f (100 µg) and control mouse IgG (100 µg) were injected i.p. three times into the mice, on days 1, 7, and 14 after HSC-2 cell injections into the mice. Tumor volume was measured on days 6, 12, 15, and 19. 5-mG2a-f-treated mice displayed significantly reduced tumor development on days 12, 15, and 19 in comparison to IgG-treated control mice (
In protocol-2 of the HSC-2 ×enograft models, tumor formation of 16 HSC-2-bearing mice was observed on day 7. Then, these 16 HSC-2-bearing mice were divided into a 5-mG2a-f-treated group and a control group. On days 7, 14, and 21 after cell injections into the mice, 5-mG2a-f (100 µg) and control mouse IgG (100 µg) were injected i.p. into the mice. Tumor volume was measured on days 7, 12, 15, 19, 22, and 27. 5-mG2a-f-treated mice displayed significantly reduced tumor development on days 22 and 27 in comparison to IgG-treated control mice (
In the present study, we investigated whether anti-CD44 mAbs are advantageous for the treatment of oral cancers. We previously developed a sensitive and specific anti-CD44 mAb, C44Mab-5, but were unable to demonstrate antitumor activity because the IgG1 subclass does not possess ADCC/CDC activities (
The most effective treatment of OSCC depends upon its clinical stage. Stage-I and -II (early stages) are treated via surgery or radiotherapy alone. In contrast, stage-III and -IV (advanced stages) require a combination of surgery, radiotherapy, and chemotherapy (
Recently, cetuximab, a mouse-human chimeric mAb (IgG1) that targets epidermal growth factor receptor (EGFR), was approved by the Food and Drug Administration (FDA) in the USA for treatment of oral cancers (
In another recent study, HER2 was shown to be expressed in oral cancers, and an anti-HER2 mAb (H2Mab-19) demonstrated antitumor activity (
Furthermore, we previously investigated whether podocalyxin (PODXL) may be a therapeutic target in OSCC using anti-PODXL mAbs (
In this study, we demonstrated that 5-mG2a-f exerts ADCC/CDC activities
In our previous report, C44Mab-5 detected CD44s (
Targeting multiple targets, such as EGFR, HER2, PODXL, and CD44 may be needed for effective therapy to conquer oral cancers. Another important goal is the targeting of cancer-specific antigens using a cancer-specific mAb (CasMab) because EGFR, HER2, PODXL, and CD44 are widely expressed in normal tissues. We previously established CasMab against podoplanin (PDPN), which is expressed in many cancers, including oral cancers (
We thank Ms. Saori Handa and Mr. Yu Komatsu (Department of Antibody Drug Development, Tohoku University Graduate School of Medicine) for technical assistance concerning the
This research was supported in part by Japan Agency for Medical Research and Development (AMED) under grant nos. JP20am0401013 (YK), JP20am0101078 (YK), and JP20ae0101028 (YK), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant nos. 17K07299 (MKK), 19K07705 (YK), and 20K16322 (MS).
The datasets used and/or analyzed during the study are available from the corresponding author on reasonable request.
HHo, JT, TO, TN, MS, TA, YS, and MY performed the experiments. MKK analyzed the experimental data. MK, HHa, and YK designed the current study and wrote the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Animal studies for ADCC and the antitumor activity were approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (Numazu-shi, Shizuoka, Japan) (permit no. 2020-003).
Not applicable.
The authors declare that they have no competing interests.
Antibody Bank of Tohoku University
antibody-dependent cellular cytotoxicity
American Type Culture Collection
bovine serum albumin
cancer-specific mAb
cell-based immunization and screening
complement-dependent cytotoxicity
CD44 standard
CD44 variant
Chinese hamster ovary
concanavalin A
Dulbecco's modified Eagle's medium
ethylenediaminetetraacetic acid
enzyme-linked immunosorbent assay
fetal bovine serum
Food and Drug Administration
fibroblast growth factor
head and neck squamous cell carcinoma
heparin-binding epidermal growth factor
Japanese Collection of Research Bioresources Cell Bank
monoclonal antibody
oral squamous cell carcinoma
phosphate-buffered saline
podoplanin
podocalyxin
polyvinylidene difluoride
receptor tyrosine kinases
reverse transcription-polymerase chain reaction
squamous cell carcinoma
sodium dodecyl sulfate
standard error of the mean
vascular endothelial growth factor receptor
Confirmation of defucosylation of 5-mG2a-f by enzyme-linked immunosorbent assay (ELISA) using lectins. (A) C44Mab-5 and 5-mG2a-f were immobilized and incubated with biotin-labeled concanavalin A (Con A), followed by peroxidase-conjugated streptavidin. The enzymatic reaction was produced using a 1-Step Ultra TMB-ELISA. (B) C44Mab-5 and 5-mG2a-f were immobilized and incubated with biotin-labeled
Flow cytometry using anti-CD44 mAbs. (A) CHO/PA16-CD44v3-10 cells were treated with C44Mab-5 and 5-mG2a-f (1 µg/ml), followed by secondary antibodies. (B) CHO-K1 cells were treated with C44Mab-5 and 5-mG2a-f (1 µg/ml), followed by secondary antibodies. (C) SAS cells were treated with C44Mab-5 and 5-mG2a-f (1 µg/ml), followed by secondary antibodies. (D) HSC-2 cells were treated with C44Mab-5 and 5-mG2a-f (1 µg/ml), followed by secondary antibodies. The black line represents the negative control. mAbs, monoclonal antibodies.
Immunohistochemical analysis using anti-CD44 mAbs against oral squamous cell carcinomas (OSCCs). (A and B) Consecutive tissue sections of OSCC were incubated with 1 µg/ml of C44Mab-5 for 1 h at room temperature followed by treatment with an Envision+ kit for 30 min. Color was developed using DAB for 2 min, and sections were then counterstained with hematoxylin. (C and D) Consecutive tissue sections of OSCC were incubated with 1 µg/ml of 5-mG2a-f for 1 h at room temperature followed by treatment with an Envision+ kit for 30 min. Color was developed using DAB for 2 min, and sections were then counterstained with hematoxylin. (E and F) Hematoxylin and eosin (HE) staining of consecutive tissue sections of OSCC. Scale bar, 100 µm. mAbs, monoclonal antibodies.
Determination of the binding affinity of anti-CD44 mAbs for oral cancer cells using flow cytometry. (A) SAS cells were suspended in 100 µl of serially diluted mAbs (0.3 ng/ml-5 µg/ml), followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were collected using an EC800 Cell Analyzer. (B) HSC-2 cells were suspended in 100 µl of serially diluted mAbs (0.3 ng/ml-5 µg/ml), followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were collected using an EC800 Cell Analyzer.
Evaluation of ADCC and CDC activities by 5-mG2a-f. (A) ADCC activities by 5-mG2a-f, control mouse IgG2a, and control PBS in SAS and HSC-2 cells. (B) CDC activities by 5-mG2a-f, control mouse IgG2a, and control PBS in SAS and HSC-2 cells. Values are mean ± SEM. Asterisk indicates statistical significance (**P<0.01; n.s., not significant; ANOVA and Tukey-Kramer's test). ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity.
Evaluation of antitumor activity of 5-mG2a-f (from day 1) in SAS xenografts. (A) SAS cells (5×106 cells) were injected subcutaneously into the left flank. After day 1, 100 µg of 5-mG2a-f and control mouse IgG in 100 µl PBS were injected i.p. into treated and control mice, respectively. Additional antibodies were then injected on days 7 and 14. Tumor volume was measured on days 6, 12, 15, and 19. Values are mean ± SEM. Asterisk indicates statistical significance (**P<0.01; n.s., not significant; ANOVA and Sidak's multiple comparisons test). (B) Tumors of SAS xenografts were resected from 5-mG2a-f and control mouse IgG groups. Tumor weight on day 19 was measured from excised xenografts. Values are mean ± SEM. Asterisk indicates statistical significance (*P<0.05, Welch's t test). (C) Resected tumors of SAS xenografts from 5-mG2a-f and control mouse IgG groups on day 19. Scale bar, 1 cm.
Evaluation of antitumor activity of 5-mG2a-f (from day 7) in SAS xenografts. (A) SAS cells (5×106 cells) were injected subcutaneously into the left flank. After day 7, 100 µg of 5-mG2a-f and control mouse IgG in 100 µl PBS were injected i.p. into treated and control mice, respectively. Additional antibodies were then injected on days 14 and 21. Tumor volume was measured on days 7, 12, 15, 19, 22, and 27. Values are mean ± SEM. Asterisk indicates statistical significance (**P<0.01; n.s., not significant; ANOVA and Sidak's multiple comparisons test). (B) Tumors of SAS xenografts were resected from 5-mG2a-f and control mouse IgG groups. Tumor weight on day 27 was measured from excised xenografts. Values are mean ± SEM. Asterisk indicates statistical significance (*P<0.05, Welch's t test). (C) Resected tumors of SAS xenografts from 5-mG2a-f and control mouse IgG groups on day 27. Scale bar, 1 cm.
Evaluation of antitumor activity of 5-mG2a-f (from day 1) in HSC-2 ×enografts. (A) HSC-2 cells (5×106 cells) were injected subcutaneously into the left flank. After day 1, 100 µg of 5-mG2a-f and control mouse IgG in 100 µl PBS were injected i.p. into treated and control mice, respectively. Additional antibodies were then injected on days 7 and 14. Tumor volume was measured on days 6, 12, 15, and 19. Values are mean ± SEM. Asterisk indicates statistical significance (**P<0.01; n.s., not significant, ANOVA and Sidak's multiple comparisons test). (B) Tumors of HSC-2 ×enografts were resected from 5-mG2a-f and control mouse IgG groups. Tumor weight on day 19 was measured from excised xenografts. Values are mean ± SEM. Asterisk indicates statistical significance (**P<0.01, Welch's t test). (C) Resected tumors of HSC-2 ×enografts from 5-mG2a-f and control mouse IgG groups on day 19. Scale bar, 1 cm.
Evaluation of antitumor activity of 5-mG2a-f (from day 7) in HSC-2 ×enografts. (A) HSC-2 cells (5×106 cells) were injected subcutaneously into the left flank. After day 7, 100 µg of 5-mG2a-f and control mouse IgG in 100 µl PBS were injected i.p. into treated and control mice, respectively. Additional antibodies were then injected on days 14 and 21. Tumor volume was measured on days 7, 12, 15, 19, 22, and 27. Values are mean ± SEM. Asterisk indicates statistical significance (**P<0.01; n.s., not significant; ANOVA and Sidak's multiple comparisons test). (B) Tumors of HSC-2 ×enografts were resected from 5-mG2a-f and control mouse IgG groups. Tumor weight on day 27 was measured from excised xenografts. Values are mean ± SEM. Asterisk indicates statistical significance (*P<0.05, Welch's t test). (C) Resected tumors of HSC-2 ×enografts from 5-mG2a-f and control mouse IgG groups on day 27. Scale bar, 1 cm.