Enhancement of tumor tropism of mPEGylated nanoparticles by anti-mPEG bispecific antibody for ovarian cancer therapy

Abstract

Ovarian cancer is highly metastatic, with a high frequency of relapse, and is the most fatal gynecologic malignancy in women worldwide. It is important to elevate the drug susceptibility and cytotoxicity of ovarian cancer cells, thereby eliminating resident cancer cells for more effective therapeutic efficacy. Here, we developed a bispecific antibody (BsAb; mPEG × HER2) that can easily provide HER2⁺ tumor tropism to mPEGylated liposomal doxorubicin (PLD) and further increase the drug accumulation in cancer cells via receptor-mediated endocytosis, and improve the cytotoxicity and therapeutic efficacy of HER2⁺ ovarian tumors. The mPEG × HER2 can simultaneously bind to mPEG molecules on the surface of PLD and HER2 antigen on the surface of ovarian cancer cells. Simply mixing the mPEG × HER2 with PLD was able to confer HER2 specificity of PLD to HER2⁺ ovarian cancer cells and efficiently trigger endocytosis and enhance cytotoxicity by 5.4-fold as compared to non-targeted PLD. mPEG × HER2-modified PLD was able to significantly increase the targeting and accumulation of HER2⁺ ovarian tumor by 220% as compared with non-targeted PLD. It could also significantly improve the anti-tumor activity of PLD (P < 0.05) with minimal obvious toxicity in a tumor-bearing mouse model. We believe that the mPEG × HER2 can significantly improve the therapeutic efficacy, potentially reduce the relapse freqency and thereby achieve good prognosis in ovarian cancer patients.

Figure 1

Characterization of mPEG × HER2. (A) The gene constructs of mPEG × HER2 and mPEG × DNS are composed of a signal peptide (S), the anti-mPEG V_L–C_κ, the anti-mPEG V_H–C_H1, a flexible linker peptide (L) and an anti-HER2 scFv (mPEG × HER2) or control anti-DNS scFv (mPEG × DNS). I, internal ribosomal entry site (IRES). The (B) mPEG and (C) HER2 binding functions of mPEG × HER2 (filled circle) or mPEG × DNS (open circle) on mPEGylated protein and HER2⁺ cancer cells were detected via ELISA (n = 2). Bars, SD. (D) The simultaneous binding ability of mPEG and HER2 antigen of mPEG × HER2 (filled circle) and mPEG × DNS (open circle) were analyzed by targeting SKOV-3 (HER2⁺) cells and detected via cell-based ELISA (n = 4). Bars, SD. The specific targeting ability of mPEG × HER2⁻ (filled circle, HER2/PLD) and mPEG × DNS-modified PLD (open circle, DNS/PLD) were analyzed by targeting (E) SKOV-3 (HER2⁺) or (F) MDA-MB-468 (HER2⁻) cells and detected via cell-based ELISA (n = 4). Bars, SD.

Figure 2

mPEG × HER2 improves the internalization of mPEGylated nanoparticles into HER2⁺ ovarian cancer cells. The internalization ability of mPEG × HER2-modified nanoparticles was performed by incubating mPEG × DNS-modified Lipo-DiD (DNS/Lipo-DiD) or mPEG × HER2-modified Lipo-DiD (HER2/Lipo-DiD) to HER2⁺ SKOV-3 cells at 4 °C for 12 h (green), 37 °C for 1 (orange), 6 (blue) and 12 h (red) and the surface bound mPEGylated nanoparticles were detected by staining with 6.3 anti-PEG antibody and FITC-conjugated secondary Ab and analyzed by flow cytometry. The internalized Lipo-DiD in SKOV-3 cells were evaluated by monitoring the (A) decreasing level of surface FITC signal. The (B) total mPEGylated nanoparticles (including surface and internalized Lipo-DiD) were monitored by detecting the red fluorescent signal of Lipo-DiD. The gray peak on the graphs show mock staining with PBS containing 0.05% (wt/vol) BSA. (C) The internalization process of mPEG × HER2-modified Lipo-DiD was investigated by confocal live cell imaging system. The HER2⁺ SKOV-3 cells were stained with Hoechst 33342 (blue), LysoTracker (green) and mPEG × DNS⁻ (DNS/Lipo-DiD) or mPEG × HER2-modified Lipo-DiD (HER2/Lipo-DiD) (red). The co-localization of Lipo-DiD with lysosome (yellow) in SKOV-3 cells was observed in real time by confocal microscopy. Scale bar 40 μm.

Figure 3

mPEG × HER2 enhances the cytotoxicity of PLD to HER2⁺ ovarian cancer through triggering apoptosis pathway. (A) PLD (filled diamond), mPEG × DNS-modified PLD (open circle, DNS/PLD) or mPEG × HER2-modified PLD (filled circle, HER2/PLD) were incubated with SKOV-3 ovarian cancer cells for 12 h. The cell viability was determined by ATPlite analysis and the mean luminescence values compared to untreated control cells (n = 3). Bars, SD. *P < 0.05. **P < 0.01. (B) mPEG × HER2, Dox, PLD, DNS/PLD or HER2/PLD were incubated with SKOV-3 ovarian cancer cells for 24 h. The apoptotic marker proteins (i.e. PARP and Caspase 9) were analyzed by Western blot and the relative ratio of apoptotic marker proteins to β-actin is shown below each lane. The cell lysates of each treated group were analyzed as described above. Lane 1, untreated group was used as a negative control. Dox, doxorubicin.

Figure 4

mPEG × HER2 improves the tumor targeting and accumulation of PLD in ovarian tumor tissue. (A) mPEG × DNS⁻ (DNS/Lipo-DiR) or mPEG × HER2-modified Lipo-DiR (HER2/Lipo-DiR) were intravenously injected in SKOV-3 tumor-bearing nude mice (red arrow at right flank). The fluorescence intensity of DiR was monitored at 24, 48 and 72 h post-injection by IVIS spectrum optical imaging system. (B) The total region of interest (ROI) in SKOV-3 tumors that were treated with mPEG × DNS⁻ (black, DNS/Lipo-DiR) or mPEG × HER2-modified Lipo-DiR (gray, HER2/Lipo-DiR) was quantified by Living Image software. (C) The total ROI of collected tumors and different organs (liver, spleen, heart, lung, ovary and kidney) treated with mPEG × DNS⁻ (black, DNS/Lipo-DiR) or mPEG × HER2-modified Lipo-DiR (gray, HER2/Lipo-DiR) were quantified at 72 h (n = 5). Bars, SEM. *P < 0.05. (D) The accumulated effect of doxorubicin of HER2-targeted PLD in ovarian tumor was measured by treating PLD, mPEG × DNS-modified PLD (DNS/PLD) or mPEG × HER2-modified PLD (HER2/PLD) to SKOV-3 tumor-bearing nude mice and detecting the fluorescent signal of doxorubicin at 48 h after treatment. The tumors were collected and the nucleus stained by DAPI. The fluorescent signal of doxorubicin (red) and DAPI (blue) were detected by confocal microscopy and the co-localized signals of doxorubicin and nucleus in HER2/PLD-treated mice are indicated (white arrow). Scale bar 40 μm.

Figure 5

mPEG × HER2 improves the therapeutic efficacy of PLD HER2-overexpressed ovarian cancer. We injected 5 mg kg⁻¹ of saline (black circle, n = 3), PLD (blue square, n = 4), mPEG × HER2 BsAb (violet circle, n = 3), mPEG × HER2⁻ (yellow square, HER2/PLD, n = 4) or mPEG × DNS-modified PLD (green square, DNS/PLD, n = 4) into SKOV-3 tumor-bearing nude mice when tumor size was 150 mm³, once a week for 4 weeks. (A) The tumor volume of each treatment and (B) the body weight were measured every 3 days post-treatment. Bars, SEM. **P < 0.01. *P < 0.05. ns not significant.