Expression of TweakR in breast cancer and preclinical activity of enavatuzumab, a humanized anti-TweakR mAb

Abstract

Background: The receptor for the cytokine TWEAK (TweakR) is a cell surface member of the tumor necrosis factor receptor superfamily with diverse biological roles. TNFRSF family members are appealing therapeutic targets in oncology due to their aberrant expression and function in tumor cells. The goal of the current study was to examine the potential of TweakR as a therapeutic target in breast cancer.

Methods: Expression of TweakR in primary breast cancer tissues and metastases was characterized using immunohistochemistry. To determine the functional relevance of TweakR, breast cancer cell lines were treated in vitro and in vivo with enavatuzumab, a humanized mAb against TweakR.

Results: Overexpression of TweakR was observed in infiltrating tumors compared to normal adjacent breast tissues, and strong staining of TweakR was observed in all subtypes of invasive ductal breast cancer. In addition, a positive correlation of TweakR and HER2 expression and co-localization were observed, irrespective of ER status. TweakR expression was also observed in bone metastasis samples from primary breast cancer but rarely in benign tumors. Enavatuzumab inhibited the in vitro growth of TweakR-expressing breast cancer cell lines, and this activity was augmented by cross-linking the mAb. In addition, enavatuzumab significantly inhibited the in vivo growth of multiple breast cancer xenograft models including a model of metastasis.

Conclusions: TweakR is highly expressed in all subtypes of invasive ductal breast cancer, and enavatuzumab administration exhibited a dose-dependent inhibition of primary tumor growth and lung metastasis and enhanced the antitumor activity of several chemotherapy agents currently used to treat breast cancer. These data provide the rationale to evaluate enavatuzumab as a potential therapy for the treatment of breast cancer.

Fig. 1

TweakR is expressed in invasive breast cancer samples and is co-expressed with HER2. Immunohistochemistry staining for TweakR expression was performed in normal adjacent tissue (a) and infiltrating tumor tissue (b) from the same case of breast cancer (200× magnification). c TweakR expression in bone metastases from breast cancer (200× magnification). d Double staining of TweakR (brown) and HER2 (Red) in a ductal breast cancer sample (400× magnification). Representative images are shown here

Fig. 2

Growth inhibition of breast cancer cell lines by enavatuzumab and synergy of inhibition when combined with trastuzumab. a–d BT549 (a), HCC38 (b), MB231 variant (c), and HCC70 (d) breast cancer cells were incubated with soluble enavatuzumab or human IgG1 control antibody in the presence (x, Δ) or absence (filled triangle, not shown) of cross-linking antibody, or immobilized enavatuzumab or control antibody (filled square, not shown) for 5–10 days. Relative viability was calculated by dividing the viability of treated cells by that of untreated cells. Representative data are shown here (point, mean of triplicate wells; bars, SEM). All experiments were repeated at least twice (E+F). SKBR3 cells were incubated with titrations of trastuzumab (e) or enavatuzumab with cross-linking secondary antibody (f) in the presence or absence of a single concentration of the other antibody (0.16 μg/mL trastuzumab and 0.26 μg/mL enavatuzumab are shown). At the effect size of 60 % inhibition (dashed line), the combination index (CI) was 0.27 for the enavatuzumab + trastuzumab combination and 0.28 for the trastuzumab + enavatuzumab combination, suggesting significant synergy between the two antibodies. Synergy was observed at the majority of antibody concentrations tested at effect sizes from 30 to 60 % inhibition (Supplemental Figure S1)

Fig. 3

Enavatuzumab inhibited the growth of primary tumors and metastases in xenograft models of breast cancer. Established MCF7 (a) and HCC70 (b) tumors were treated with enavatuzumab or a human IgG1 control antibody at 10 mg/kg three times per week, with 10 animals in each dosing group. Tumor volumes were measured on each dosing day; points, mean; bars, SEM. Differences in tumor volumes between the enavatuzumab and control treated groups were significant (p < 0.001) in both models. c–e Established MB231 variant tumors were treated with enavatuzumab or a human IgG1 control antibody thrice per week, with 7 mice in each dosing group. c Tumor volumes were measured on each dosing day; points, mean; bars, SEM. d Metastases were quantified in lungs harvested on day 37; both micrometastases (<4 cells) and metastatic clusters >4 cells were enumerated. Enavatuzumab treatment significantly inhibited the growth of primary tumors and micrometastases at 10, 3, and 1 mg/kg and inhibited the development of larger metastases at 10 and 3 mg/kg (*p < 0.05; **p < 0.001). e Immunohistochemical staining of human cytokeratin-positive metastases in mouse lungs

Fig. 4

Enavatuzumab enhanced the antitumor activity of gemcitabine and navelbine in a triple-negative model of breast cancer. Established MB231 variant xenograft tumors were treated with enavatuzumab at 1 mg/kg thrice weekly, alone or in combination with gemcitabine at 30 mg/kg (a) or vinorelbine at 5 mg/kg (b) twice weekly, with 7 mice in each dosing group. Tumor volumes were measured on gemcitabine and vinorelbine dosing days; points, mean; bars, SEM. Differences in tumor volumes were significant (p < 0.05) between the gemcitabine and enavatuzumab/gemcitabine treatment groups and the vinorelbine and enavatuzumab/vinorelbine treatment groups on days 22–34. The data shown in a and b were derived from a single study. A second study yielded similar results