Development of an Antibody Delivery Method for Cancer Treatment by Combining Ultrasound with Therapeutic Antibody-Modified Nanobubbles Using Fc-Binding Polypeptide

Abstract

A key challenge in treating solid tumors is that the tumor microenvironment often inhibits the penetration of therapeutic antibodies into the tumor, leading to reduced therapeutic efficiency. It has been reported that the combination of ultrasound-responsive micro/nanobubble and therapeutic ultrasound (TUS) enhances the tissue permeability and increases the efficiency of delivery of macromolecular drugs to target tissues. In this study, to facilitate efficient therapeutic antibody delivery to tumors using this combination system, we developed therapeutic antibody-modified nanobubble (NBs) using an Fc-binding polypeptide that can quickly load antibodies to nanocarriers; since the polypeptide was derived from Protein G. TUS exposure to this Herceptin^®-modified NBs (Her-NBs) was followed by evaluation of the antibody's own ADCC activity, resulting the retained activity. Moreover, the utility of combining therapeutic antibody-modified NBs and TUS exposure as an antibody delivery system for cancer therapy was assessed in vivo. The Her-NBs + TUS group had a higher inhibitory effect than the Herceptin and Her-NBs groups. Overall, these results suggest that the combination of therapeutic antibody-modified NBs and TUS exposure can enable efficient antibody drug delivery to tumors, while retaining the original antibody activity. Hence, this system has the potential to maximize the therapeutic effects in antibody therapy for solid cancers.

Keywords: Fc-binding polypeptide; antibody delivery; cancer; nanobubbles; ultrasound.

Figure 1

Schematic drawing of the structure of therapeutic antibody-modified nanobubbles (NBs) and the in vivo mechanism of action for the combination of therapeutic antibody-modified NBs and therapeutic ultrasound (TUS) exposure. Therapeutic antibody-modified NBs, which are loaded with Fc-binding polypeptide, expand to a much larger size upon TUS exposure and then collapse. The formation of jets caused by the collapse of NBs perforates surrounding blood vessels and membranes, enhancing the therapeutic antibody accumulation in tumor tissues.

Figure 2

Characterization of non-modified PEG-NBs and Her-NBs. (A) Particle size distribution was measured via Laser Diffraction Method. (B) Attachment of Her-NBs and cancer cells. DiI-labeled NBs were incubated with SKOV3 (HER2⁺ cancer) cells or MDA-MB-231 (HER2⁻ cancer) cells for 5 min, and then DAPI was added to the cells to stain nuclei. Red: DiI-labeled NBs, Blue: DAPI, scale bar: 50 µm. (C) Ultrasound contrast images of NBs before/after therapeutic ultrasound (TUS) exposure (frequency, 1 MHz; duty, 50%; intensity, 1 W/cm²; time, 2 min).

Figure 3

Activity of Herceptin on Her-NBs before/after TUS exposure. To evaluate the activity of Herceptin, luminescence intensity of Jurkat effector cells, expressing NFAT-RE-luc2, was measured by ADCC reporter bioassay against SKOV3 (T). SKOV3 cells were plated in a 96-well plate the before assay. On the day of assay, samples and effector cells (E) were added to the plate (cell rate; E:T = 15:1). After 6 h of incubation at 37 °C/5% CO₂ condition, Bio-Glo^TM luciferase assay regent was added, and the luminescence intensity was determined using luminometer. (A) Luminescence intensity of Herceptin in Her-NBs after TUS in serial concentration of lipid. (B) Comparison of luminescence intensity of Her-NBs before and after TUS exposure in 2.5 µg of lipid. (C) Calculation of titer of Herceptin in Her-NBs using the standard curve of the amount of Herceptin versus luminescence intensity. Data = mean ± SD (n = 4). The unpaired t-test was used for statistical analysis.

Figure 3

Activity of Herceptin on Her-NBs before/after TUS exposure. To evaluate the activity of Herceptin, luminescence intensity of Jurkat effector cells, expressing NFAT-RE-luc2, was measured by ADCC reporter bioassay against SKOV3 (T). SKOV3 cells were plated in a 96-well plate the before assay. On the day of assay, samples and effector cells (E) were added to the plate (cell rate; E:T = 15:1). After 6 h of incubation at 37 °C/5% CO₂ condition, Bio-Glo^TM luciferase assay regent was added, and the luminescence intensity was determined using luminometer. (A) Luminescence intensity of Herceptin in Her-NBs after TUS in serial concentration of lipid. (B) Comparison of luminescence intensity of Her-NBs before and after TUS exposure in 2.5 µg of lipid. (C) Calculation of titer of Herceptin in Her-NBs using the standard curve of the amount of Herceptin versus luminescence intensity. Data = mean ± SD (n = 4). The unpaired t-test was used for statistical analysis.

Figure 4

Ultrasonography images of Her-NBs with/without TUS groups in tumors in vivo. SKOV3-bearing mice (Tumor volume: approximately 200–300 mm³) were injected with Her-NBs intravenously, and contrast images of the tumors were captured. The tumor was exposed to TUS (frequency, 1 MHz; duty, 50%; intensity, 1 W/cm²; time, 2 min) immediately after injection in the TUS-exposure groups. Contrast images of (A) Her-NBs without TUS from 0 min to 5 min and (B) Her-NBs with TUS at 0 min and immediately after TUS exposure. Images were captured by US diagnostic machine with a 12 MHz probe. The circles show the area of the tumor. Scale bar: 5 mm.

Figure 5

In vivo efficacy of the combination of Her-NBs and TUS exposure against SKOV3 tumor models. Tumor growth rate (A) and changes in body weight (B) on day 8. Tumor-bearing mice (tumor volume: approximately 65 mm³) were injected with PBS, Herceptin (0.8 μg/head), or NBs three times every other day. The black arrows indicate treatment days. The tumor was exposed to TUS (frequency, 1 MHz; duty, 50%; intensity, 1 W/cm²; time, 2 min) immediately after injection in the TUS-exposure groups. Relative tumor volumes and relative body weights are expressed as multiples of the initial volumes on day 1. Data = mean ± SD (n = 4). * p < 0.05 (one-way ANOVA followed by the Tukey’s test).