Biochemical and Biophysical Research Communications
A bispecific anti-ErbB2 antibody potently induces ErbB2 internalization and suppresses ErbB2-overexpressing tumor growth
Introduction
ErbB2 (also known as Her2 or Neu) is a member of the ErbB family of receptor tyrosine kinases, which also includes ErbB1 (or EGFR), ErbB3 and ErbB4 [1]. ErbB2 is the preferred heterodimerization partner of the other ErbB family members and could enhance their signaling, although ErbB2 has no known ligand of its own [2], [3]. It is found overexpressed in approximately 25%–30% of human breast cancers [4], [5], and about 4%–50% of human gastric cancers [6]. Surface overexpression of ErbB2 drives tumorigenesis, cancer progression and poor prognosis [7], [8]. Thus, ErbB2 is an important immunogenic and is an ideal candidate for antibody-based cancer immunotherapy.
Anti-ErbB2 monoclonal antibodies (mAbs) are shown to improve the survival of advanced gastric cancer patients that harboring ErbB2 overexpression and are currently also explored in adjuvant and neoadjuvant settings [6], [9]. The molecular mechanisms underlying the growth-inhibitory effects of anti-ErbB2 monoclonal antibodies may involve antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), increased cancer cell apoptosis, as well as direct interference with signaling cascades [10]. Down-regulation and endocytosis of ErbB2 are also widely believed to be an important mechanism [11], [12], [13], [14]. Previous studies have related enhanced endocytosis of ErbB2 to reduced tumorigenesis [15], [16]. Endocytosis and degradation of ErbB2 may contribute to the blockade of growth-promoting signaling pathways, resulting in ErbB2-driven cancer cell growth inhibition [17].
Ben-Kasus T and his colleagues have found that as long as two anti-ErbB2 antibodies did not interfere with each other, the combination of them would synergistically induce ErbB2 internalization [18], [19], [20], [21], [22], [23]. Here, we explored the ability of 18 combinations of two non-competitive ErbB2-specific mAbs to induce ErbB2 internalization. To our surprise, most of the non-competitive mAb combinations induced ErbB2 internalization. Interestingly, although the non-competitive anti-ErbB2 mAbs trastuzumab and pertuzumab, either alone or in combination, were ineffective at inducing ErbB2 internalization, a bispecific antibody engineered from these two antibodies potently down-regulated the ErbB2 molecule. The antitumor effect of this anti-ErbB2 bispecific antibody was also investigated.
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Cell lines and animals
The human gastric cancer cell line NCI-N87 was purchased from the American Type Culture Collection (ATCC). The cell line was authenticated twice by morphologic and isoenzyme analyses during the study period. Cell line was routinely checked for contamination by mycoplasma using Hoechst staining and consistently found to be negative. Four-week-old female BALB/c nude mice were obtained from the Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China). All animals were
Characterization of epitopes recognized by anti-ErbB2 monoclonal antibodies
It has been reported that trastuzumab binds to the juxtamembrane region of domain IV of ErbB2, and pertuzumab recognizes domain II. Here, we developed 5 mouse anti-human ErbB2 mAbs 1H3, 3B5, 7C6, 10F1 and 12D4. Competitive binding assay results showed that all of the five mouse mAbs did not interfere with the binding of trastuzumab and pertuzumab (Fig. 1 and Table 1). The binding of 7C6 to ErbB2 was not affected by all the other anti-ErbB2 mAbs tested. The mouse mAb 3B5, but not 7C6, 10F1 and
Discussion
Recent studies have addressed an emerging strategy that could enhance the therapeutic activity of antireceptor antibodies by combining two mAbs engaging distinct epitopes. And the synergistic antigrowth effects of antibody combinations are partly mediated by Abs-induced internalization and degradation of surface receptors [20], [21], [29]. Endocytosis of surface receptors may lead to down-regulation of downstream growth-promoting signals, thus increasing antitumor efficacy of Abs.
Conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
This work was supported by National Natural Science Foundation of China (81572996), Shanghai Key Laboratory of Cell Engineering (14DZ2272300) and Shanghai Leading Academic Discipline Project (B905).
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These authors contributed equally to this work.