A Novel Anti-HER2 Bispecific Antibody With Potent Tumor Inhibitory Effects In Vitro and In Vivo

Abstract

Overexpression of HER2 has been reported in many types of cancer, making it a perfect candidate for targeted immunotherapy. The combination of two FDA approved monoclonal antibodies (mAbs), trastuzumab and pertuzumab, has more robust anti-tumor activity in patients with HER2-overexpressing breast cancer. We recently produced a new humanized anti-HER2 mAb, hersintuzumab, which recognizes a different epitope than trastuzumab and pertuzumab on HER2. This mAb, in combination with trastuzumab, exhibits more potent anti-tumor activity than each parental mAb alone. Here we have developed a novel bispecific anti-HER2 antibody (BsAb) designated as trasintuzumab, composed of trastuzumab and hersintuzumab, using dual variable domain immunoglobulin (DVD-Ig) technology. Both variable domains of trasintuzumab are fully functional and have similar affinities to the parental mAbs and are also able to bind to natural HER2 on the surface of several HER2-expressing cell lines. Trasintuzumab was found to inhibit the growth of different types of tumor cell lines through suppression of the AKT and ERK signaling pathways as efficiently as the combination of the parental mAbs. It also induced tumor regression as potently as the combination of the two mAbs in nude mice bearing ovarian and gastric cancer xenografts. Our data suggest that trasintuzumab may be a promising BsAb therapeutic candidate for the treatment of HER2-overexpressing cancers.

Keywords: DVD-Ig; HER2; bispecific antibody; cancer immunotherapy; monoclonal antibody.

Figure 1

Structural characterization of anti-HER2 DVD-Igs. (A) Schematic presentation of BiHT and BiTH design. (B) SDS-PAGE analysis of trastuzumab, BiHT and BiTH under reducing (left) and non-reducing (right) conditions. Tra, trastuzumab; Her, hersintuzumab; HC, heavy chain; LC, light chain; MS, molecular size.

Figure 2

The profile of binding of DVD-Igs and their parental mAbs to HER2 and its subdomains. Different concentrations of antibodies were comparatively determined by an immunoglobulin-specific ELISA (A). Binding patterns of trastuzumab, hersintuzumab, BiHT, and BiTH with full HER2-ECD and its subdomains DI+II and DIII+IV were analyzed by a specific ELISA (B–D).

Figure 3

Binding competition and related IC50 of BsAbs. Trastuzumab, hersintuzumab, BiHT, and BiTH compete with trastuzumab-HRP (A, C) or hersintuzumab-HRP (B, D) for binding to recombinant HER2-ECD (A, B), and subdomains III+IV (C) and I+II (D) by ELISA. Binding competition of trastuzumab, hersintuzumab, BiHT, and BiTH with FITC-labeled trastuzumab (E) and FITC-labeled hersintuzumab (F) to native HER2 on SKOV-3 cell line was also assessed by flow cytometry. IC50: The half maximal inhibitory concentration.

Figure 4

Binding profile of DVD-Igs and their parental antibodies to HER2-expressing tumor cell lines. BT-474, JIMT-1, HCC-1954, MCF-7, SKOV-3, and NCI-N87 cell lines were treated with trastuzumab, hersintuzumab, BiHT, BiTH as primary antibodies and then FITC labeled anti-human Ig polyclonal antibody was used as the secondary antibody. The final results were analyzed by flow cytometry. A chimeric anti-hepatitis B mAb harboring human IgG1/κ was used as isotype control.

Figure 5

Anti-proliferative effects of BsAbs and their parental mAbs on the proliferation of BT-474, HCC-1954, JIMT-1, SKOV-3, and NCI-N87 cell lines. Data represent percentage of cell growth inhibition of triplicate cultures conducted three times. Combination denotes treatment with trastuzumab and hersintuzumab at a half dose of the indicated concentration for each mAbs. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns: non-significant.

Figure 6

Antibody dependent cell cytotoxicity (ADCC) of the BsAbs. SKOV-3 cells were cocultured with human PBMCs in the prescence of serial concentrations of mAbs and LDH release was measured in the supernatants. A chimeric anti-hepatitis B mAb harboring human IgG1/κ was used as isotype negative control. The ADCC experiments were performed in triplicate, and the data are representative of mean ± SEM of two independent experiments. EC50: The half maximal effective concentration, HB: hepatitis B, **P < 0.01.

Figure 7

Pharmacokinetics profile of BiHT, trastuzumab, and hersintuzumab. Serum concentrations (± SEM) were measured at different time intervals after administration of a single dose of 10 mg/kg (intraperitoneally/IP or intravenously/IV) in BALB/c mice (3 mice/group). IP: intraperitoneal, IV: intravenous.

Figure 8

Inhibitory effects of BiHT on phosphorylation of the AKT and ERK1/2 signaling pathways and HER2 expression. BT-474, SKOV-3, NCI-N87, HCC-1954, JIMT-1, and MCF-7 cell lines were treated with BiHT (20 µg/ml) or combination of trastuzumab and hersintuzumab (10 µg/ml, each) for 24 h. Cell lysates were separated on SDS-PAGE and immunoblotted to detect AKT, pAKT, ERK1/2, pERK1/2, HER2, and beta actin as a house keeping protein. Cnt: Control, Com: Combination. The figures presented underneath of each mAb treatment represent the band densities calculated as described in Materials and Methods.

Figure 9

In vivo therapeutic efficacy of BiHT mAb in nude mice. SKOV-3 (A) and NCI-N87 (B) xenograft tumor bearing nude mice were treated once a week with a combination of trastuzumab plus hersintuzumab (5 mg/kg of each), or BiHT (10 mg/kg), for 6 consecutive weeks and the control group received excipient alone. Tumor sizes were measured three times a week. Data is shown as mean ± SEM. ****P < 0.0001. Tra: trastuzumab. Her: hersintuzumab. Arrows show timing of mAb administration.