A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8+ T cells.

Strategies to increase intratumoral concentrations of an anticancer agent are desirable to optimize its therapeutic potential when said agent is efficacious primarily within a tumor but also have significant systemic side effects. Here, we generate a bifunctional protein by fusing interleukin-10 (IL-10) to a colony-stimulating factor-1 receptor (CSF-1R)-blocking antibody. The fusion protein demonstrates significant antitumor activity in multiple cancer models, especially head and neck cancer. Moreover, this bifunctional protein not only leads to the anticipated reduction in tumor-associated macrophages but also triggers proliferation, activation, and metabolic reprogramming of CD8+ T cells. Furthermore, it extends the clonotype diversity of tumor-infiltrated T cells and shifts the tumor microenvironment (TME) to an immune-active state. This study suggests an efficient strategy for designing immunotherapeutic agents by fusing a potent immunostimulatory molecule to an antibody targeting TME-enriched factors.

Ferroptosis Signature Shapes the Immune Profiles to Enhance the Response to Immune Checkpoint Inhibitors in Head and Neck Cancer.

As a type of immunogenic cell death, ferroptosis participates in the creation of immunoactive tumor microenvironments. However, knowledge of spatial location of tumor cells with ferroptosis signature in tumor environments and the role of ferroptotic stress in inducing the expression of immune-related molecules in cancer cells is limited. Here the spatial association of the transcriptomic signatures is demonstrated for ferroptosis and inflammation/immune activation located in the invasive front of head and neck squamous cell carcinoma (HNSCC). The association between ferroptosis signature and inflammation/immune activation is more prominent in HPV-negative HNSCC compared to HPV-positive ones. Ferroptotic stress induces PD-L1 expression through reactive oxygen species (ROS)-elicited NF-κB signaling pathway and calcium influx. Priming murine HNSCC with the ferroptosis inducer sensitizes tumors to anti-PD-L1 antibody treatment. A positive correlation between the ferroptosis signature and the active immune cell profile is shown in the HNSCC samples. This study reveals a subgroup of ferroptotic HNSCC with immune-active signatures and indicates the potential of priming HNSCC with ferroptosis inducers to increase the antitumor efficacy of immune checkpoint inhibitors.

Loss of cell-cell adhesion triggers cell migration through Rac1-dependent ROS generation.

Epithelial cells usually trigger their "migratory machinery" upon loss of adhesion to their neighbors. This default is important for both physiological (e.g., wound healing) and pathological (e.g., tumor metastasis) processes. However, the underlying mechanism for such a default remains unclear. In this study, we used the human head and neck squamous cell carcinoma (HNSCC) SAS cells as a model and found that loss of cell-cell adhesion induced reactive oxygen species (ROS) generation and vimentin expression, both of which were required for SAS cell migration upon loss of cell-cell adhesion. We demonstrated that Tiam1-mediated Rac1 activation was responsible for the ROS generation through NADPH-dependent oxidases. Moreover, the ROS-Src-STAT3 signaling pathway that led to vimentin expression was important for SAS cell migration. The activation of ROS, Src, and STAT3 was also detected in tumor biopsies from HNSCC patients. Notably, activated STAT3 was more abundant at the tumor invasive front and correlated with metastatic progression of HNSCC. Together, our results unveil a mechanism of how cells trigger their migration upon loss of cell-cell adhesion and highlight an important role of the ROS-Src-STAT3 signaling pathway in the progression of HNSCC.

Sumoylation participates in the regulation of YB-1-mediated mismatch repair deficiency and alkylator tolerance.

Numerous reports indicate that enhanced expression of Y-box binding protein-1 (YB-1) in tumor cells is strongly associated with tumorigenesis, aggressiveness, drug resistance, as well as poor prognosis in several types of cancers, and YB-1 is considered to be an oncogene. The molecular mechanism contributing to the regulation of the biological activities of YB-1 remains obscure. Sumoylation, a post-translational modification involving the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to a target protein, plays key roles in the modulation of protein functions. In this study, our results revealed that YB-1 is sumoylated and that Lys26 is a critical residue for YB-1 sumoylation. Moreover, YB-1 was found to directly interact with SUMO proteins, and disruption of the SUMO-interacting motif (SIM) of YB-1 not only interfered with this interaction but also diminished YB-1 sumoylation. The subcellular localization, protein stability, and transcriptional regulatory activity of YB-1 were not significantly affected by sumoylation. However, decreased sumoylation disrupted the interaction between YB-1 and PCNA as well as YB-1-mediated inhibition of the MutSα/PCNA interaction and MutSα mismatch binding activity, indicating a functional role of YB-1 sumoylation in inducing DNA mismatch repair (MMR) deficiency and spontaneous mutations. The MMR machinery also recognizes alkylator-modified DNA adducts to signal for cell death. We further demonstrated that YB-1 sumoylation is crucial for the inhibition of SN1-type alkylator MNNG-induced cytotoxicity, G2/M-phase arrest, apoptosis, and the MMR-dependent DNA damage response. Collectively, these results provide molecular explanations for the impact of YB-1 sumoylation on MMR deficiency and alkylator tolerance, which may provide insight for designing therapeutic strategies for malignancies and alkylator-resistant cancers associated with YB-1 overexpression.

Polysaccharide-containing fraction from Artemisia argyi inhibits tumor cell-induced platelet aggregation by blocking interaction of podoplanin with C-type lectin-like receptor 2.

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism that involves the protection of tumor cells in the circulation and the promotion of tumor cell invasion and metastases. The C-type lectin-like receptor 2 (CLEC-2) that binds podoplanin (PDPN) is on the platelet surface and facilitates the TCIPA. Selective blockage of the PDPN-mediated platelet-tumor cell interaction is thereby a plausible strategy for inhibiting metastases. In a search for antagonists of PDPN- and tumor cell-induced platelet aggregation, traditional Chinese medicines were screened and it was found that the water extract of Artemisia argyi leaves selectively inhibited the PDPN-induced platelet aggregation. Bioactivity-guided fractionation analysis was performed for defining a polysaccharide-containing fraction (AAWAP) characterized by inhibition of PDPN activity and tumor cell-induced platelet aggregation. The pharmacological effects of AAWAP on PDPN-activated CLEC-2 signaling were determined by using Western blot and alpha screening analyses. AAWAP was non-toxic to the cells and platelets and it suppressed PDPN- and tumor cell-induced platelet aggregation by irreversibly blocking the interaction between PDPN and CLEC-2 in a dose-dependent manner. These findings indicate that AAWAP is an antagonist of the PDPN-CLEC-2 interaction. This action by AAWAP may result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.

Multiview Contouring for Breast Tumor on Magnetic Resonance Imaging.

The shape and contour of the lesion are shown to be effective features for physicians to identify breast tumor as benign or malignant. The region of the lesion is usually manually created by the physician according to their clinical experience; therefore, contouring tumors on breast magnetic resonance imaging (MRI) is difficult and time-consuming. For this purpose, an automatic contouring method for breast tumors was developed for less burden in the analysis and to decrease the observed bias to help in making decisions clinically. In this study, a multiview segmentation method for detecting and contouring breast tumors in MRI was represented. The preprocessing of the proposed method reduces any amount of noises but preserves the shape and contrast of the breast tumor. The two-dimensional (2D) level-set segmentation method extracts contours of breast tumors from the transverse, coronal, and sagittal planes. The obtained contours are further utilized to generate appropriate three-dimensional (3D) contours. Twenty breast tumor cases were evaluated and the simulation results show that the proposed contouring method was an efficient method for delineating 3D contours of breast tumors in MRI.

Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377-RGDSFTHTPP-386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma.

Podocalyxin (PODXL) overexpression is associated with progression, metastasis, and poor outcomes in cancers. We recently produced the novel anti-PODXL monoclonal antibody (mAb) PcMab-47 (IgG1, kappa). Herein, we engineered PcMab-47 into 47-mG2a, a mouse IgG2a-type mAb, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed 47-mG2a-f, a core fucose-deficient type of 47-mG2a to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and 47-mG2a detected PODXL in 163/201 (81.1%) and in 197/201 (98.0%) OSCC samples, respectively. 47-mG2a-f also detected PODXL in OSCCs at a similar frequency as 47-mG2a. In vitro analysis revealed that both 47-mG2a and 47-mG2a-f exhibited strong complement-dependent cytotoxicity (CDC) against CHO/hPODXL cells. In contrast, 47-mG2a-f exhibited much stronger ADCC than 47-mG2a against OSCC cells, indicating that ADCC and CDC of those anti-PODXL mAbs depend on target cells. In vivo analysis revealed that both 47-mG2a and 47-mG2a-f exerted antitumor activity in CHO/hPODXL xenograft models at a dose of 100 µg or 500 µg/mouse/week administered twice. 47-mG2a-f, but not 47-mG2a, exerted antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 µg/mouse/week administered three times. Although both 47-mG2a and 47-mG2a-f exerted antitumor activity in HSC-2 xenograft models at a dose of 500 µg/mouse/week administered twice, 47-mG2a-f also showed higher antitumor activity than 47-mG2a. These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs.

Epitope Mapping of Monoclonal Antibody PMab-48 Against Dog Podoplanin.

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on normal renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells. Increased expression of PDPN in cancers is associated with poor prognosis and hematogenous metastasis through interactions with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously reported a novel PMab-48 antibody, which is an anti-dog PDPN (dPDPN) monoclonal antibody (mAb) recognizing PDPN expressed in lymphatic endothelial cells. However, the binding epitope of PMab-48 is yet to be clarified. In this study, an enzyme-linked immunosorbent assay and flow cytometry were used to investigate epitopes of PMab-48. The results revealed that the critical epitope of PMab-48 comprises Asp29, Asp30, Ile31, Ile32, and Pro33 of dPDPN.

Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L1Mab-13 (IgG1, kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L1Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L1Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L1Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

Podoplanin: An emerging cancer biomarker and therapeutic target.

Podoplanin (PDPN) is a transmembrane receptor glycoprotein that is upregulated on transformed cells, cancer associated fibroblasts and inflammatory macrophages that contribute to cancer progression. In particular, PDPN increases tumor cell clonal capacity, epithelial mesenchymal transition, migration, invasion, metastasis and inflammation. Antibodies, CAR-T cells, biologics and synthetic compounds that target PDPN can inhibit cancer progression and septic inflammation in preclinical models. This review describes recent advances in how PDPN may be used as a biomarker and therapeutic target for many types of cancer, including glioma, squamous cell carcinoma, mesothelioma and melanoma.

Epitope Mapping of Monoclonal Antibody PMab-52 Against Cat Podoplanin.

The mucin-type membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in numerous malignant cancers, including squamous cell carcinoma, germinal neoplasia, mesothelioma, lung cancer, oral cancer, and brain tumor. PDPN expression is strongly associated with cancer progression and poor prognosis. Furthermore, PDPN binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation to facilitate tumor metastasis. We have previously reported a novel anti-cat PDPN (cPDPN) monoclonal antibody (mAb), PMab-52, which specifically detects cPDPN using flow cytometry analysis and successfully identifies cPDPN in feline squamous cell carcinomas. However, the specific binding epitope of cPDPN for PMab-52 remains unelucidated. In this study, a series of deletion or point mutants of cPDPN were utilized for investigating the binding epitopes of PMab-52 using flow cytometry and Western blotting. The findings of this study revealed that the critical epitopes of platelet aggregation-stimulating domain 4 (PLAG4) of cPDPN are responsible for the binding of PMab-52 to cPDPN.

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H2Mab-119 (IgG1, kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H2Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H2Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H2Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H2Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

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.

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, activates downstream signaling cascades in many tumors. In this study, we established novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We immunized mice with a combination of the extracellular domain of EGFR and EGFR-overexpressing LN229 glioblastoma cells (LN229/EGFR) and performed the first screening using enzyme-linked immunosorbent assay. Next, we selected mAbs using flow cytometry. Among 156 established clones, two mAbs, EMab-51 (IgG1, kappa) and EMab-134 (IgG1, kappa), reacted with EGFR in Western blot analysis; EMab-134 showed a much higher sensitivity compared with EMab-51. We compared the binding affinities of EMab-51 and EMab-134 using flow cytometry; the calculated KD values for EMab-51 and EMab-134 against SAS cells/HSC-2 cells were 9.2 × 10-9 M/9.9 × 10-9 M and 2.6 × 10-9 M/8.3 × 10-9 M, respectively, indicating that EMab-134 has a higher affinity to EGFR-expressing cells. Immunohistochemical analysis of EMab-51 and EMab-134 showed sensitive and specific reactions against oral cancer cells; EMab-134 demonstrated a much higher sensitivity (36/38 cases; 94.7%) to oral squamous cell carcinomas compared with EMab-51 (6/38 cases; 15.8%). This novel anti-EGFR mAb, EMab-134, could be advantageous for detecting EGFR in the pathological analysis of EGFR-expressing cancers.

Epitope Mapping of Monoclonal Antibody PMab-38 Against Dog Podoplanin.

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is extensively expressed by normal lymphatic endothelial cells, renal podocytes, and pulmonary type I alveolar cells. Nevertheless, increased expression of PDPN in malignant tumors not only associates with poor prognosis but also facilitates hematogenous metastasis through interaction with C-type lectin-like receptor-2 presented on platelets, followed by PDPN-mediated platelet activation. We previously reported a novel PMab-38 antibody, an anti-dog PDPN (dPDPN) monoclonal antibody, which specifically recognizes PDPN in squamous cell carcinomas melanomas and cancer-associated fibroblasts in canine cancer tissues. However, the specific binding with the epitope of PMab-38 remains undefined. In this study, flow cytometry was utilized to investigate the epitope of PMab-38, which was determined using a series of deletion or point mutants of dPDPN. The results revealed that the critical epitope of PMab-38 is Tyr67 and Glu68 of dPDPN.

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

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. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG1, kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry.

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG2a, kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

Antipodocalyxin Antibody chPcMab-47 Exerts Antitumor Activity in Mouse Xenograft Models of Colorectal Adenocarcinomas.

Podocalyxin (PODXL) is expressed in several cancers, including brain tumors and colorectal cancers. PODXL overexpression is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human PODXL, which was produced using LN229 glioblastoma cells, and produced a clone PcMab-47 that could be used for investigating PODXL expression by flow cytometry and immunohistochemical analysis. Herein, we produced a human-mouse chimeric PcMab-47 (chPcMab-47) and investigated its antitumor activity against PODXL-expressing tumors. chPcMab-47 reacted with LN229, LN229/PODXL, and Chinese hamster ovary (CHO)/PODXL cells, but it did not react with CHO-K1 or PODXL-knockout LN229 cell line (PDIS-13). chPcMab-47 exerted antitumor activity against a mouse xenograft model using CHO/PODXL. Furthermore, chPcMab-47 was reactive with colorectal cancer cell lines such as HCT-15, Caco-2, HCT-8, and DLD-1. chPcMab-47 also exhibited antitumor activity against a mouse xenograft model using HCT-15. These results suggest that chPcMab-47 could be useful for antibody therapy against PODXL-expressing cancers.

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H2Mab-77 (IgG1, kappa) was selected. Finally, immunohistochemical analyses with H2Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H2Mab-77 to detect HER2 in pathological analyses of breast cancers.