Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer

Introduction

Human epidermal growth factor receptor 2 (HER2) has been reported to be overexpressed in >20% of patients with breast cancer and is associated with poor clinical outcomes.^(1,2) Trastuzumab and pertuzumab, humanized anti-HER2 monoclonal antibodies (mAbs), and trastuzumab emtansine, an antibody-drug conjugate, have been approved for the treatment of HER2-positive breast cancer.^(3–5) Trastuzumab treatment has resulted in significant survival benefits for patients with metastatic HER2-positive breast cancer.⁽⁶⁾ Furthermore, the combination of pertuzumab and trastuzumab with chemotherapy has led to significant improvements in overall survival compared with trastuzumab alone plus chemotherapy.⁽⁷⁾ In addition, HER2 overexpression has been reported in gastric cancers,⁽⁸⁾ lung cancers,⁽⁹⁾ pancreatic cancers,⁽¹⁰⁾ and colorectal cancers.⁽¹¹⁾ In this study, we developed a novel anti-HER2 mAb and characterized it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses.

Materials and Methods

Results and Discussion

We immunized mice with the purified recombinant extracellular domain of HER2. We performed flow cytometry to check reactions with LN229 and LN229/HER2 cells. A stronger reaction against LN229/HER2 was needed compared with LN229 because LN229 cells endogenously express HER2. We obtained one clone: H₂Mab-139 (IgG₁, kappa). Flow cytometry analysis revealed that H₂Mab-139 reacted with LN229/HER2 cells more strongly than with endogenous HER2-expressing LN229 glioblastoma cells (Fig. 1A). H₂Mab-139 also reacted with Chinese hamster ovary (CHO)/HER2 cells but did not react with CHO-K1 cells (data not shown), indicating that H₂Mab-139 is specific for HER2. Western blot analysis of H₂Mab-139 against LN229 and LN229/HER2 cells revealed that the molecular weight of H₂Mab-139 was ∼200 kDa in LN229/HER2 cells (Fig. 1B).

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FIG. 1.

Characterization of H₂Mab-139. (A) Flow cytometry with H₂Mab-139; cells were treated with 10 μg/mL of H₂Mab-139, followed by Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control. (B) Western blot using H₂Mab-139; cell lysates were electrophoresed, and proteins were transferred onto PVDF membranes. After blocking, the membranes were incubated with 10 μg/mL of H₂Mab-139 and 1 μg/mL of anti-β-actin (AC-15) and subsequently incubated with peroxidase-conjugated anti-mouse IgG. (C) Flow cytometry with H₂Mab-139; colon cancer cell lines were treated with 10 μg/mL of H₂Mab-139, followed by Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control. (D) Sections of colon cancer were incubated with 10 μg/mL of H₂Mab-139 for 1 hour at room temperature, followed by treatment with Envision+ kit for 30 minutes. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB) for 2 minutes; subsequently, sections were counterstained with hematoxylin. Scale bar = 100 μm. HER2, human epidermal growth factor receptor 2; H₂Mab-139, anti-HER2 monoclonal antibody; PVDF, polyvinylidene difluoride.

H₂Mab-139 recognized endogenous HER2 in colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1 (Fig. 1C). However, H₂Mab-139 did not detect HER2 in colon cancer cell lines in western blots (data not shown), indicating that H₂Mab-139 is not suitable for western blot analysis of colon cancer cell lines.

We further investigated the immunohistochemical utility of H₂Mab-139 in human colon cancers. Figure 1D shows that H₂Mab-139 stained the cell membranes of colon cancers that were previously diagnosed as HER2-positive. H₂Mab-139 also stained the cell membranes of HER2-positive breast cancer cells (Supplementary Fig. S1), indicating that H₂Mab-139 is useful for the immunohistochemical analysis for HER2-positive cancers. In the future, we intend to determine the positive rate of many colon cancer tissues via immunohistochemical analysis using H₂Mab-139.