Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti-HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2-positive breast and gastric cancer. An anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa) was previously developed. In the present study, a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding-affinity [a dissociation constant (
Human epidermal growth factor receptor 2 (HER2, also known as ERBB2) is a cell surface type I transmembrane glycoprotein that is highly expressed on various solid tumors and enable a broad repertoire of oncogenic signaling upon homo- and heterodimerization with HER/ERBB families. HER2 overexpression is observed in ~20-30% of human breast cancers, which are associated with poor prognosis and higher rates of recurrence (
Trastuzumab was initially considered to inhibit HER2 signaling (
Trastuzumab possesses an Fc domain which allows for the direct engagement with Fcγ receptors (FcγRs) on various types of immune cells. The FcγR engagement allows for phagocytic engulfment of antibody-bound pathogens or cells, termed antibody-dependent cellular phagocytosis. The FcγR-mediated signaling activates dendritic cells, macrophages and neutrophils, which can alter adaptive immune responses through antigen presentation, cytokine production and chemotaxis. Furthermore, the FcγR engagement can stimulate natural killer (NK) cells which attack and lyse the target cells, termed antibody-dependent cellular cytotoxicity (ADCC) (
With the increase in lifespan of both humans and dogs, the increased cancer incidence has been observed as well. Mammary neoplasia is the most frequently observed in dog tumors (
Previously, an anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa), was developed (
A canine mammary gland tumor cell line, SNP, was purchased from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer at Tohoku University (Miyagi, Japan) (
Animal experiments were performed following regulations and guidelines to minimize animal distress and suffering in the laboratory. Animal experiments for antitumor activity of H77Bf were approved (approval no. 2021-056) by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (Numazu, Japan). Mice were maintained on an 11 h light/13 h dark cycle with food and water supplied
Anti-HER2 mAb H2Mab-77 was established as previously described (
CHO-K1, CHO/dHER2, and SNP were harvested by 0.25% trypsin/1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.) treatment. After washing with blocking buffer [0.1% bovine serum albumin (BSA; Nacalai Tesque, Inc.) in phosphate-buffered saline (PBS)], cells were treated with H77Bf, or blocking buffer (control) for 30 min at 4°C. Then, cells were incubated in FITC-conjugated anti-dog IgG (cat. no. A18764; 1:1,000; Thermo Fisher Scientific, Inc.) for 30 min at 4°C. Fluorescence data were collected by the Cell Analyzer EC800 and analyzed by EC800 software ver. 1.3.6 (Sony Corp.).
CHO/dHER2 and SNP were suspended in serially diluted H77Bf (0.006–25 µg/ml) followed by FITC-conjugated anti-dog IgG (1:200). Fluorescence data were collected using the Cell Analyzer EC800. The dissociation constant (
Cells were fixed with 4% paraformaldehyde-PBS for 10 min and quenched with 50 mM NH4Cl in PBS with 0.2 mM Ca2+ and 2 mM Mg2+. The cells were blocked with blocking buffer (PBS containing 0.2 mM Ca2+, 2 mM Mg2+ and 0.5% BSA) for 30 min and incubated with 10 µg/ml of H77Bf or blocking buffer for 1 h. The cells were further incubated with Alexa Fluor 488-conjugated anti-dog IgG (1:400; Jackson ImmunoResearch Laboratories, Inc.) and 0.3 µM of 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Inc.) for 45 min. The whole processes were performed at room temperature. Fluorescence images were acquired with a 40× objective on a BZ-X800 digital fluorescence microscope (Keyence Corporation).
Canine mononuclear cells (MNCs) obtained from Yamaguchi University were resuspended in DMEM (Nacalai Tesque, Inc.) with 10% FBS and were used as effector cells (
Cytolyticity (% lysis) was calculated as follows: % lysis=(E-S)/(M-S) ×100, where ‘E’ is the fluorescence in cultures of both effector and target cells, ‘S’ is the spontaneous fluorescence of only target cells, and ‘M’ is the maximum fluorescence following the treatment with a lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM of EDTA, and 0.5% Triton X-100).
Target cells (CHO-K1, CHO/dHER2, and SNP) were labeled with 10 µg/ml Calcein AM (
BALB/c nude mice (female, 5 weeks old, weighing 14–17 g) were purchased from Charles River Laboratories, Inc. CHO-K1, CHO/dHER2, or SNP cells (5×106 cells) were resuspended in DMEM and mixed with BD Matrigel Matrix Growth Factor Reduced (BD Biosciences) were subcutaneously injected into the left flank of mice.
On day 8 post-inoculation, 100 µg of H77Bf (n=8) or control dog IgG (n=8) in 100 µl PBS were intraperitoneally injected. On days 14 and 21, additional antibody inoculations were performed. Furthermore, on days 8, 14 and 21, canine MNCs were injected surrounding the tumors. The tumor volume was measured on days 7, 10, 14, 17, 21, 24 and 28 after the injection of cells. Tumor volumes were determined as previously described (
All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was conducted with Welch's t test for ADCC, CDC, and tumor weight. ANOVA with Sidak's post hoc test were conducted for tumor volume and mouse weight. All calculations were performed using GraphPad Prism 8 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
In our previous study, an anti-HER2 mAb (H2Mab-77) was established using cancer-specific mAb (CasMab) method (
A kinetic analysis of the interactions of H77Bf with CHO/dHER2 cells was performed via flow cytometry. As revealed in
It was examined whether H77Bf is applicable for immunocytochemistry. The H77Bf specificity was evaluated by using CHO/dHER2 and CHO-K1 cells. As revealed in
It was investigated whether H77Bf was capable of mediating ADCC against CHO/dHER2 cells. H77Bf showed ADCC (31.8% cytotoxicity) against CHO/dHER2 cells more effectively than the control dog IgG (13.2% cytotoxicity; P<0.05). There was no difference between H77Bf and control dog IgG about ADCC against CHO-K1 (
It was then examined whether H77Bf could exert CDC against CHO/dHER2 cells. As revealed in
In the CHO/dHER2 ×enograft tumor, H77Bf and control dog IgG were intraperitoneally injected into mice on days 8, 14 and 21, following the CHO/dHER2 cells injection. On days 7, 10, 14, 17, 21, 24 and 28 after the injection, the tumor volume was measured. The H77Bf administration resulted in a significant reduction of tumors on days 24 (P<0.01) and 28 (P<0.01) compared with that of the control dog IgG (
The weight of CHO/dHER2 tumors treated with H77Bf was significantly lower than that treated with control dog IgG (71% reduction; P<0.05;
In the CHO-K1 ×enograft models, H77Bf and control dog IgG were injected intraperitoneally into mice on days 8, 14 and 21 after the injection of CHO-K1 cells. The tumor volume was measured on days 7, 10, 14, 17, 21, 24 and 28 after the injection of cells. No difference was observed between H77Bf and control dog IgG about CHO-K1 tumor volume (
The body weights loss and skin disorder were not observed in CHO/dHER2 (
As demonstrated in
Immunocytochemical analysis was then performed using H77Bf for SNP cells. As a result, H77Bf detected dHER2 on SNP cells (
It was investigated whether H77Bf was capable of mediating ADCC against SNP cells. As revealed in
In the SNP xenograft models, H77Bf and control dog IgG were injected intraperitoneally on days 8, 14 and 21, after the injection of SNP cells. The tumor volume was measured on days 7, 10, 14, 17, 21, 24 and 28 after the injection. The H77Bf administration resulted in a significant reduction in tumor growth on days 10 (P<0.01), 14 (P<0.01), 17 (P<0.01), 21 (P<0.01), 24 (P<0.01) and 28 (P<0.01) compared with that of the control dog IgG (
Tumors from the H77Bf-treated mice weighed significantly less than those from the control dog IgG-treated mice (35% reduction; P<0.05,
The body weights loss and skin disorder were not observed in SNP tumor-bearing mice (
Human mAbs that exhibit cross-reactivity to dog have been investigated. It has been suggested that cetuximab (anti-EGFR) and trastuzumab (anti-HER2) can bind to certain canine cancer cell lines (
Drug-conjugated mAbs rely on direct cytotoxicity of the payloads through endocytosis of receptor-bound mAbs-drug conjugate (
IHC has played a critical role as a diagnostic tool for the identification of neoplasms with conventional histopathology. In human breast cancer pathology, IHC is routinely used to assist with the prognosis and to determine the specific treatment (e.g. trastuzumab) for patients. Although IHC is not routinely used in CMTs, an increasing number of studies have been looking for reliable diagnostic and/or prognostic IHC biomarkers including dHER2 (
The authors would like to thank Ms. Miyuki Yanaka, Mr. Takuro Nakamura, Mr. Yu Komatsu and Ms. Saori Handa (Department of Antibody Drug Development, Tohoku University Graduate School of Medicine) for technical assistance of
All data generated or analyzed during this study are included in this published article.
TO, TT, MS and TA performed the experiments. MKK, MK and YK designed the experiments. TM prepared canine MNCs. TA, HS, TY and YK analyzed the data. HS and YK wrote the manuscript. All authors read and approved the final 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.
The animal study protocol was approved (approval no. 2021-056) by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (Numazu, Japan).
Not applicable.
The authors declare that they have no competing interests.
human epidermal growth factor receptor 2
monoclonal antibody
antibody-dependent cellular cytotoxicity
complement-dependent cytotoxicity
Food and Drug Administration
phosphatidylinositol-3 kinase
tyrosine kinase inhibitor
Fcγ, receptor
natural killer
canine mammary tumor
Roswell Park Memorial Institute
phosphate-buffered saline
dissociation constant
4′,6-diamidino-2-phenylindole
mononuclear cell
standard error of the mean
immunohistochemistry
Trastuzumab deruxtecan
Flow cytometry using H77Bf. (A) Production of H77Bf (core-fucose-deficient dog IgGB) from H2Mab-77 (mouse IgG1). (B) CHO-K1 and CHO/dHER2 cells were treated with H77Bf or buffer control, followed by FITC-conjugated anti-dog IgG. (C) Determination of the binding affinity of H77Bf using flow cytometry for CHO/dHER2 cells. CHO/dHER2 cells were suspended in serially diluted H77Bf, followed by the addition of FITC-conjugated anti-dog IgG. Fluorescence data were analyzed using the EC800 Cell Analyzer. (D) Immunocytochemistry using H77Bf. CHO-K1 and CHO/dHER2 cells were incubated with buffer control or 10 µg/ml H77Bf for 1 h, followed by the incubation with Alexa Fluor 488-conjugated anti-dog IgG and DAPI for 45 min. Fluorescent images were acquired using a fluorescent microscope BZ-X800. Scale bars, 20 µm.
Evaluation of ADCC and CDC elicited by H77Bf. (A) ADCC elicited by H77Bf and control dog IgG targeting CHO/dHER2 and CHO-K1 cells. (B) CDC elicited by H77Bf and control dog IgG targeting CHO/dHER2 and CHO-K1 cells Values are presented as the mean ± SEM. (*P<0.05; Welch's
Antitumor activity of H77Bf. (A and B) Evaluation of tumor volume in (A) CHO/dHER2 and (B) CHO-K1 ×enograft models. CHO/dHER2 and CHO-K1 cells (5×106 cells) were subcutaneously injected into mice. On day 8, 100 µg of H77Bf or control dog IgG were injected intraperitoneally into mice. Additional antibodies were injected on days 14 and 21. Mononuclear cells were also injected surrounding the tumors on days 8, 14 and 21. The tumor volume was measured on days 7, 10, 14, 17, 21, 24 and 28 after the injection. Values are presented as the mean ± SEM. **P<0.01 (ANOVA and Sidak's multiple comparisons test). (C and D) Tumor weight (day 28) was measured from excised xenografts of (C) CHO/dHER2 and (D) CHO-K1. Values are presented as the mean ± SEM. *P<0.05 (Welch's
Body weights and appearance of the mice. (A and B) Body weights of mice implanted with (A) CHO/dHER2 and (B) CHO-K1 ×enografts on days 7, 10, 14, 17, 21, 24 and 28 (ANOVA and Sidak's multiple comparisons test). (C and D) Body appearance of (C) CHO/dHER2 and (D) CHO-K1-implanted mice on day 28 (scale bar, 1 cm). n.s., not significant.
Flow cytometry, ADCC and CDC activity of H77Bf against canine mammary gland tumor cell line, SNP cells. (A) SNP cells were treated with H77Bf or buffer control, followed by FITC-conjugated anti-dog IgG. (B) Determination of the binding affinity of H77Bf for SNP cells using flow cytometry. SNP cells were suspended in 100 µl of serially diluted H77Bf, followed by the addition of FITC-conjugated anti-dog IgG. Fluorescence data were collected using the EC800 Cell Analyzer. (C) Immunocytochemistry using H77Bf. SNP cells were incubated with buffer control or 10 µg/ml H77Bf for 1 h, followed by the incubation with Alexa Fluor 488-conjugated anti-dog IgG and DAPI for 45 min. Fluorescent images were acquired using a fluorescent microscope BZ-X800 (scale bars, 20 µm). (D) ADCC and CDC elicited by H77Bf and control dog IgG targeting SNP cells. Values are presented as the mean ± SEM. *P<0.05; Welch's t-test). ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity.
Antitumor activity of H77Bf against SNP xenograft. (A) Evaluation of tumor volume in SNP xenograft models. SNP cells (5×106 cells) were injected subcutaneously into mice. On day 8, 100 µg of H77Bf or control dog IgG in 100 µl PBS were injected intraperitoneally into mice. Additional antibodies were injected on days 14 and 21. Mononuclear cells were also injected surrounding the tumors on days 8, 14 and 21. The tumor volume was measured on days 7, 10, 14, 17, 21, 24 and 28 after the inoculation. Values are presented as the mean ± SEM. **P<0.01 (ANOVA and Sidak's multiple comparisons test). (B) Tumor weight (day 28) was measured from excised SNP xenografts. Values are presented as the mean ± SEM. *P<0.05 (Welch's
Body weights and appearance of the mice. (A) Body weights of mice implanted with SNP xenografts on days 7, 10, 14, 17, 21, 24 and 28 (ANOVA and Sidak's multiple comparisons test). (B) Body appearance of SNP-implanted mice on day 28 (scale bar, 1 cm). n.s., not significant.