In vivo activity of novel anti-ErbB2 antibody chA21 alone and with Paclitaxel or Trastuzumab in breast and ovarian cancer xenograft models

Abstract

It was well studied that ErbB2 (HER2/p185(her2/neu)) overexpression in human malignant cancers correlates with poor prognosis and chemo-resistance. Although Trastuzumab (Herceptin) has been widely used in patients with ErbB2-overexpressing metastatic breast cancer, many patients either do not respond to Trastuzumab therapy or progress within 1 year of initiating Trastuzumab treatment. Previously, we reported a novel tumor-inhibitory antibody chA21, which recognized ErbB2 extracellular domain with an epitope distinct from other tumor-inhibitory anti-ErbB2 antibodies. Here, we report that chA21 combined with Paclitaxel or Trastuzumab significantly enhances the tumor-inhibition effects on ErbB2-overexpressing breast and ovarian cancer in xenograft mice. Moreover, the study reveals that the effects by chA21 to cause an enhanced inhibition on cancer cell proliferation and angiogenesis was highly associated with the intrinsic ability of chA21 to down-regulate ErbB2 receptor, inhibit downstream MAPK and PI3K-AKT signal transduction and activate natural killer cells. Our findings show that chA21 may represent a unique anti-ErbB2 antibody with potentials as therapeutic candidate alone or combination with other anti-ErbB2 reagents in cancer therapy.

Fig. 1

Growth inhibition on tumor xenografts by chA21. a ErbB2-positive expression on BT-474 and SKOV-3 tumor xenografts confirmed by IHC method. Scale bar 500 μm. b A dose-dependent response induced by chA21 on both BT-474 and SKOV-3 xenografts. chA21 was given i.v. twice a week for total 24 days at doses of 20, 30 and 40 mg/kg. *p < 0.01 compared with control. **p < 0.01 compared with 20 mg/kg chA21 group. Results are given as representative of two independent experiments. c Dose-dependent down-regulation of ErbB2 receptor on both BT-474 and SKOV-3 xenografts by chA21. By the end of in vivo experiment, tumors were excised, lysed and subjected to western blots with specific antibodies against ErbB2 ECD (Ab-20) and GAPDH

Fig. 2

Enhanced anti-tumor efficacies by chA21 in combination with Paclitaxel or Trastuzumab. The mice bearing SKOV-3 or BT-474 xenografts were given i.v. by normal saline, 30 mg/kg chA21 twice a week, 10 mg/kg Paclitaxel once a week, 20 mg/kg Trastuzumab twice a week, or both combination with chA21 for total 28 days. *p < 0.01 compared with control. **p < 0.01 compared with individual drug group. Tra Trastuzumab, PTX Paclitaxel. Results are given as representative of two independent experiments

Fig. 3

Enhanced inhibition of cell proliferation and angiogenesis by chA21 in combination with Paclitaxel or Trastuzumab. a HE and IHC staining showing pathological changes on cell proliferation and angiogenesis of BT-474 xenografts. Light-colored (red) arrows show blood vessels, and dark-colored (black) arrows denote cell vacuolization. The image scale bars of HE, Ki-67 and VEGF are 50 μm, and that of CD31 is 500 μm. Representative images are shown. b Quantification of Ki-67-, VEGF-positive cells and MVD by CD31 staining of BT-474 xenografts. The number of positive cells or MVD was shown as the percent of average number versus control. *p < 0.01 compared with control. **p < 0.01 compared with the individual drug group. Tra Trastuzumab, PTX Paclitaxel. Similar results were obtained on SKOV-3 xenografts

Fig. 4

Down-regulation of ErbB2 receptor and interruption of downstream signaling by chA21. a At the end of in vivo experiments, tumors were lysed and 50 μg of total protein from each sample was loaded on SDS-PAGE. Then the levels of p185ErBb2 and p95ErBb2 and downstream signal proteins were analyzed by western blot with specific antibodies as described in “Materials and methods”. GAPDH was used as loading control. Tra Trastuzumab, PTX Paclitaxel. b The bands were scanned by densitometry and analyzed using ImageJ 1.42. The ratios of phopsho-AKT to AKT (p-AKT/AKT) and phopsho-ERK to ERK (p-ERK/ERK) were calculated and the values were shown as ratios calculated according to the control treatment, which was arbitrarily taken as 1.0. Tra Trastuzumab, PTX Paclitaxel. *p < 0.01 compared with control. **p < 0.01 compared with chA21, Paclitaxel or Trastuzumab alone. Similar results were obtained on SKOV-3 xenografts

Fig. 5

Increased number and activation of NK cells by chA21. a Nude mice bearing BT-474 xenografts were treated i.v. with indicated drug alone or combinations. At the end of in vivo experiment, the total numbers of NK cells (DX5⁺ CD3⁻ cells) in peripheral blood and the percentages of NK cells in lymphocytes were determined by flow cytometry. b NK cells were evaluated for CD69 and NKG2D expression by flow cytometry. Histograms show CD69 and NKG2D expression on this cell subset, and mean fluorescence intensity (MFI) denotes expression level. Tra Trastuzumab, PTX Paclitaxel. Results were presented as the average ± SD. Similar results were obtained in mice bearing SKOV-3 xenografts. c Immunohistochemical staining for NK cells was performed on the paraffin-embedded tumor sections. A scale bar in each panel represents 200 μm. d The number of immunopositive cells per field were determined as described in “Materials and methods” and were presented as the average ± SD calculated from determinations of seven tumor samples of each group. *p < 0.001 compared with control. **p < 0.001 compared with the individual drug group. Tra Trastuzumab, PTX Paclitaxel. Similar results were obtained on SKOV-3 xenografts