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. 2016 Jan 1;6(1):118-30.
doi: 10.7150/thno.13069. eCollection 2016.

Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors

Shou-Cheng Wu  1 , Yu-Jen Chen  1 , Hsiang-Ching Wang  2 , Min-Yuan Chou  2 , Teng-Yuan Chang  3 , Shyng-Shiou Yuan  4 , Chiao-Yun Chen  5 , Ming-Feng Hou  6 , John Tsu-An Hsu  7 , Yun-Ming Wang  8
Affiliations

Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors

Shou-Cheng Wu et al. Theranostics. .

Abstract

The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR.

Keywords: EGFR.; HER2/neu; MnMEIO; bispecific antibody; magnetic resonance imaging.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Fig 1
Fig 1
Schematic diagram of tetravalent bispecific antibody. (A) Structure of scFv and human Fc domains, and (B) assembled tetravalent bispecific antibody. Purification and structural characterization of tetravalent bispecific antibody (Bis). (C) Confirmation of tetravalent bispecific antibody expression by Western blot analysis. (D) Purified tetravalent bispecific antibody were electrophoresed on SDS-PAGE under non-reducing condition (DTT (-)) and reducing conditions (DTT (+)).
Fig 2
Fig 2
TEM images and schematic representation of MnMEIO NPs conjugated with (right) or without (left) mPEG, CyTE777 and the bispecific antibody. The core size of MnMEIO NPs was 9.1 ± 0.3 nm. Then, the organic and aqueous dispersions of nanoparticles were also shown.
Fig 3
Fig 3
Binding activity of MnMEIO-FITC-(Bis)-mPEG NPs and the control NPs (MnMEIO-FITC-mPEG (Blank), MnMEIO-FITC-(Her)-mPEG, MnMEIO-FITC-(Erb)-mPEG, and MnMEIO-FITC-(Erb+Her)-mPEG NPs) to (A) SKBR-3, (B) A431, and (C) Colo-205 cells. In these experiments NPs (10 µg/mL) were incubated with cells for 1 hr at 37 °C. Cell-associated fluorescent intensities were analyzed with a FACScan flow cytometer Binding activity of MnMEIO-FITC-(Bis)-mPEG NPs and the control NPs (MnMEIO-FITC-mPEG (Blank), MnMEIO-FITC-(Her)-mPEG, MnMEIO-FITC-(Erb)-mPEG, and MnMEIO-FITC-(Erb+Her)-mPEG NPs) to (A) SKBR-3, (B) A431, and (C) Colo-205 cells. In these experiments NPs (10 µg/mL) were incubated with cells for 1 hr at 37 °C. Cell-associated fluorescent intensities were analyzed with a FACScan flow cytometer.
Fig 4
Fig 4
In vitro optical imaging of SKBR-3, A431, and Colo-205 tumor cells after the treatment with MnMEIO-CyTE777-(Bis)-mPEG NPs and control NPs (MnMEIO-CyTE777 -mPEG, MnMEIO-CyTE777-(Her)-mPEG, MnMEIO-CyTE777-(Erb)-mPEG, and MnMEIO-CyTE777-(Erb+Her)-mPEG NPs). In these experiments NPs (10 μg/mL) were incubated with cells for 1 hr at 37 °C. (A) Fluorescence images in a multi-well format were obtained by the IVIS spectrum system; (B) Quantitative analysis of fluorescence intensities of (A).
Fig 5
Fig 5
The in vitro T2-weighted images of SKBR-3, A431, and Colo-205 tumor cells after treatment with MnMEIO-CyTE777-(Bis)-mPEG NPs and control NPs (MnMEIO-CyTE777 -mPEG, MnMEIO-CyTE777-(Her)-mPEG, MnMEIO-CyTE777-(Erb)-mPEG, and MnMEIO-CyTE777-(Erb+Her)-mPEG NPs). In these experiments, NPs (10 µg/mL) were incubated with cells for 1 hr at 37 °C. MR imaging was performed with a 7.0 T MR imaging scanner.
Fig 6
Fig 6
Confocal microscopy images of SKBR-3, A431, and Colo-205 tumor cells incubated with MnMEIO-FITC-(Bis)-mPEG NPs (10 µg/mL) for 1 hr at 37 °C. HiLyte FluorTM 594 stained the cytoplasm in red and DAPI stained the nuclei in blue.
Fig 7
Fig 7
(A) Pharmacokinetics of MnMEIO-CyTE777-(Bis)-mPEG and control NPs (MnMEIO-CyTE777-mPEG NPs) in nude mice bearing subcutaneous tumor xenografts of SKBR-3 and Colo-205 tumor cells. The blood clearances were compared at pre-injection and various time points (2, 5, 10, 30, 60, 180, 360, 720, and 1440 mins) post-injection; (B) iron concentrations upon various time points based on (A).
Fig 8
Fig 8
(A) In vivo optical images of nude mice bearing subcutaneous SKBR-3 and Colo-205 tumor xenografts after intravenous injection of MnMEIO-CyTE777-mPEG and MnMEIO-CyTE777-(Bis)-mPEG NPs (10 mg/kg); (B) quantitative fluorescent intensities of the NPs in the tumors with images acquired at pre-injection and various time points (2, 8, 24, and 96 hrs) post-injection; (C) in vivo white light and near-infrared fluorescence images of dissected organs of mice bearing SKBR-3 and Colo-205 tumors sacrificed after 96 hrs post-injection of MnMEIO-CyTE777-(Bis)-mPEG NPs (excitation: 745 nm; emission: 820 nm). (1) lipid, (2) liver, (3) heart, (4) kidney, (5) stomach, (6) lung, (7) spleen, (8) intestines, (9) Colo-205 tumor, and (10) SKBR-3 tumor; (D) quantification of fluorescence intensities of isolated organs.
Fig 9
Fig 9
T2-weighted MR images (7.0 T) of nude mice bearing subcutaneous SKBR-3 and Colo-205 tumor xenografts before (Pre), and 2 hrs and 24 hrs after injection of MnMEIO-CyTE777-(Bis)-mPEG NPs (10 mg/kg).
Fig 10
Fig 10
Histological analyses of (A) Colo-205 and (B) SKBR-3 tumor tissue specimens acquired 96 hrs after injection of MnMEIO-CyTE777-(Bis)-mPEG NPs (10 mg/kg). Sections were stained with Prussian blue. The accumulation of MnMEIO-CyTE777-(Bis)-mPEG NPs is indicated by arrows.
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