Preparation and evaluation of the PD0721‑DOX antibody‑drug conjugate targeting EGFRvIII to inhibit glioblastoma

Hu,Minmin; Liu,Hong; Zhang,Yubing; Lu,Dingyan; Zheng,Lin; Wang,Yonglin; Chen,Shuaishuai; Liu,Ting

doi:10.3892/etm.2024.12542

Preparation and evaluation of the PD0721‑DOX antibody‑drug conjugate targeting EGFRvIII to inhibit glioblastoma

Authors:
- Minmin Hu
- Hong Liu
- Yubing Zhang
- Dingyan Lu
- Lin Zheng
- Yonglin Wang
- Shuaishuai Chen
- Ting Liu
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Affiliations: State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Provincial Key Laboratory of Pharmaceutics, Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China, Engineering Research Center for The Development and Application of Ethnic Medicine and TCM (Ministry of Education)/National Engineering Research Center of Miao's Medicines, Guizhou Medical University, Guiyang, Guizhou 550004, P.R. China
Published online on: April 17, 2024 https://doi.org/10.3892/etm.2024.12542
Article Number: 254
Copyright: © Hu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Epidermal growth factor receptor variant III (EGFRvIII) is prominently expressed in various epithelial tumors. PD0721, a single‑chain antibody (scFv), has been developed to specifically target EGFRvIII. Although doxorubicin (DOX) is an essential treatment approach for glioblastoma (GBM), its toxic effects and limited targeting capabilities are a challenge. To overcome the above limitations, antibody‑drug conjugates (ADCs) have been developed to exploit the specificity of monoclonal antibodies in directing potent cytotoxic drugs to tumor cells expressing the target antigens. The present study aimed to conjugate DOX with PD0721 scFv to construct a PD0721‑DOX ADC targeting EGFRvIII and examine its targeting effect and in vitro anti‑GBM activity. PD0721‑DOX ADC was generated by combining PD0721 scFv with DOX, using dextran T‑10 as a linker. The drug‑to‑antibody ratio (DAR) was measured by ultraviolet and visible spectrophotometry (UV‑Vis). A series of techniques, including cytotoxicity assays, immunofluorescence, cell internalization and flow cytometry assays were employed to evaluate the targeting efficacy and anti‑GBM activity of the PD0721‑DOX ADC. Following the conjugation of PD0721 scFv with DOX, the UV‑Vis results showed a noticeable red shift in the maximum absorbance. The DAR of PD0721 scFv and DOX was 9.23:1. Cytotoxicity assays demonstrated that DK‑MG cells treatment with PD0721‑DOX ADC at 10 and 20 µg/ml significantly increased cytotoxicity compared with U‑87MG ATCC cells (all P<0.01). Confocal microscopy revealed distinct green and red fluorescence in EGFRvIII‑expressing DK‑MG cells, while no fluorescence was observed in EGFRvIII negative U‑87MG ATCC cells. Furthermore, compared with U‑87MG ATCC cells, DK‑MG cells showed effective internalization of the PD0721‑DOX ADC (P<0.001). Finally, flow cytometric analyses indicated that the PD0721‑DOX ADC significantly promoted the apoptosis of DK‑MG cells compared with U‑87MG ATCC cells (P<0.01). In summary, the current study suggested that the PD0721‑DOX ADC could exhibit a notable targeting efficacy and potent anti‑GBM activity.

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