Anti-Metastatic Activity of an Anti-EGFR Monoclonal Antibody against Metastatic Colorectal Cancer with KRAS p.G13D Mutation

Abstract

The now clinically-used anti-epidermal growth factor receptor (EGFR) monoclonal antibodies have demonstrated significant efficacy only in patients with metastatic colorectal cancer (mCRC), with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS). However, no effective treatments for patients with mCRC with KRAS mutated tumors have been approved yet. Therefore, a new strategy for targeting mCRC with KRAS mutated tumors is desired. In the present study, we examined the anti-tumor activities of a novel anti-EGFR monoclonal antibody, EMab-17 (mouse IgG_2a, kappa), in colorectal cancer (CRC) cells with the KRAS p.G13D mutation. This antibody recognized endogenous EGRF in CRC cells with or without KRAS mutations, and showed a high sensitivity for CRC cells in flow cytometry, indicating that EMab-17 possesses a high binding affinity to the endogenous EGFR. In vitro experiments showed that EMab-17 exhibited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities against CRC cells. In vivo analysis revealed that EMab-17 inhibited the metastases of HCT-15 and HCT-116 cells in the livers of nude mouse metastatic models, unlike the anti-EGFR monoclonal antibody EMab-51 of subtype mouse IgG₁. In conclusion, EMab-17 may be useful in an antibody-based therapy against mCRC with the KRAS p.G13D mutation.

Keywords: antibody-dependent cell cytotoxicity; colorectal cancer; complement-dependent cytotoxicity; epidermal growth factor receptor; metastasis.

Figure 1

Flow cytometry of epidermal growth factor receptor (EGFR)-transfected and EGFR-expressing cell lines using anti-EGFR antibodies EMab-17 and EMab-51. (A) CHO-K1 and CHO/EFGR cells were subjected to Western blotting analysis with an anti-EGFR or anti-β-tubulin antibody. (B) CHO-K1 and EGFR-transfected CHO-K1, CHO/EGFR cells were treated with 1 μg/mL of anti-EGFR antibodies, EMab-51 (Left; blue) and EMab-17 (Right; red). After treatment with the anti-EGFR antibodies, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control. Fluorescence data were collected using a Cell Analyzer EC800. (C) CRC cell lines with or without KRAS mutations were treated with EMab-51 (Top; blue) and EMab-17 (Bottom; red). Yellow highlights indicate the KRAS status of CRC cell lines. Data were analyzed as in (B).

Figure 2

Antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and anti-tumor activities of EMab-17 against CHO/EGFR cells. (A) ADCC (Left) and CDC (Right) activities of EMab-17 against CHO-K1 (Top) or CHO/EGFR (Bottom) in vitro. ADCC activity was evaluated through the calcein-AM release assay in the presence of antibodies (100 μg/mL; effector/target ratio, 50). CDC activity was determined using the MTS assay in the presence of antibodies (100 μg/mL) or control mouse IgG_2a (100 μg/mL) with 10% rabbit complement. **: p < 0.01 vs. IgG_2a-treated control. (B, E) Anti-tumor activity of EMab-17 against CHO-K1 or CHO/EGFR cells in vivo. Tumor volume of CHO-K1 (B) or CHO/EGFR (E) xenografts. Cells (5 × 10⁶ cells/100 μL) were subcutaneously inoculated into BALB/c nude mice. After 1 day, 100 μg of EMab-17 or control mouse IgG were injected into the peritoneal cavities of the mice. The orange arrows indicate the days of antibody injection and the antibodies were injected thrice (days 1, 7 and 14; control: n = 8; EMab-17: n = 8). The tumor diameter was measured at the indicated days and calculated using the formula: tumor volume = W² × L/2, where W is the short diameter and L is the long diameter. Values are presented as means (SD). n.s.: not significant, **: p < 0.01 vs. control. (C, F) Resected tumors of CHO-K1 (C) or CHO/EGFR (F) xenografts on day 21. Scale bar: 10 mm. (D, G) Tumor weight of CHO-K1 (D) or CHO/EGFR (G) xenografts. Values are presented as means ± SEM. n.s.: not significant, **: p < 0.01 vs. control.

Figure 3

EMab-17 inhibits the growth of colorectal cancer (CRC) cell lines with KRAS p.G13D mutation. (A) Anti-tumor activity of EMab-17 against HCT-15 (Top) and HCT-116 (Bottom) cells in vivo. Tumor volume of xenografts treated with mouse control IgG, EMab-17 or EMab-51. Cells (5 × 10⁶ cells/100 μL) were subcutaneously inoculated into BALB/c nude mice. After 1 day, 100 μg of EMab-51, EMab-17 or control mouse IgG were injected into the peritoneal cavities of the mice. The orange arrows indicate the days of antibody injection and the antibodies were injected thrice (days 1, 7, and 15; control: n = 8; EMab-51: n = 8, EMab-17: n = 8). Values are presented as means (SD). *: p < 0.05, **: p < 0.01 vs. control. (B) Resected tumors of HCT-15 or HCT-116 xenografts on day 18. (C) Tumor weight of HCT-15 (Left) or HCT-116 (Right) xenografts. Scale bar: 1 cm. Values are presented as means ± SEM. **: p < 0.01 vs. control and EMab-51-treated groups.

Figure 4

Determination of the binding affinity and ADCC and CDC activities of EMab-17 or EMab-51 using flow cytometry. (A) HCT-15 (Top) and HCT-116 (Bottom) cells were suspended in 100 μL of serially diluted EMab-17 or EMab-51 (6 to 100 ng/mL), followed by the addition of secondary anti-mouse IgG. Fluorescence data were collected using a cell analyzer. (B) The ADCC and CDC activities of EMab-17 or EMab-51 against HCT-15 (Top) or HCT-116 (Bottom) cells were evaluated. ADCC activity was evaluated through the calcein-AM release assay in the presence of antibodies (100 μg/mL; effector/target ratio, 12.5, 25 and 50). CDC activity was determined using the MTS assay in the presence of antibodies (100 μg/mL) or control mouse IgGs (IgG₁ or IgG_2a, 100 μg/mL) with 10% rabbit complement. The values are means (SD). *: p < 0.05, **: p < 0.01 vs. IgG_2a-treated control.

Figure 5

Characterization of a model of CRC metastasis to the liver in nude mice. (A) The experimental schedule. Cloned GFP-labeled HCT-15 (HCT-15-GFP) cells (2.5 × 10⁵ cells or 5 × 10⁵ cells suspended with Matrigel/10 μL) were injected into the spleens. Mice were sacrificed 27 days after the injection of cancer cells, and the livers were removed to determine the frequency of liver metastasis. (Upper right) The pictures of cloned HCT-15-GFP cells. Scale bar: 50 μm. (B) Growth of HCT-15-GFP cells in the injected spleens and formation of metastasis in livers. A total of 2.5 × 10⁵ cells (left) or 5 × 10⁵ cells (right) were injected into the spleen, and images were captured after sacrifice. Representative images of body (a, g), spleen (b, i), liver (c, d, h), abdominal cavity (e, j) and lung (f). The arrows point to the site of metastasis. Scale bar: 10 mm. (C) The status of the model is shown in the table. The number of metastatic lesions in the liver is shown as means ± SEM. The frequency of liver metastasis and tumor growth in the spleen are shown in brackets. (D) Representative histological appearance of metastasized HCT-15-GFP cells in the liver (left) and immunohistochemical detection of HCT-15-GFP cells. Scale bar: 100 μm.

Figure 6

Anti-metastatic effects of an anti-EGFR antibody EMab-17 on CRC metastasis to the liver. (A) HCT-15-GFP and cloned GFP-labeled HCT-116 (HCT-116-GFP) cells were subjected to Western blotting analysis with an anti-EGFR, anti-GFP or anti-β-tubulin antibody. (B) Flow cytometry using the anti-EGFR antibody EMab-17 for HCT-15-GFP or HCT-116-GFP cells. (C) Experimental schedule. HCT-15-GFP and HCT-116-GFP cells (2.5 × 10⁵ cells suspended with Matrigel/10 μL) were injected into the spleens. The antibodies (100 μg/mouse) were intraperitoneally administered 1, 10, 17 and 23 days after the inoculation of cancer cells. The mice were sacrificed 26 or 27 days after the injection of cancer cells, and the livers were removed to determine the number of metastatic CRC cells. (D) Representative images of the liver. The arrows point to the site of metastasis. Scale bar: 1 cm. (E) Number of metastatic tumors in the liver. The mean number for each group is expressed with a horizontal bar; 10 mice were used per group. The values are presented as means ± SEM. *: p < 0.05 vs. control and EMab-51-treated groups.