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An ultra-small bispecific protein augments tumor penetration and treatment for pancreatic cancer

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European Journal of Nuclear Medicine and Molecular Imaging Aims and scope Submit manuscript

A Correction to this article was published on 01 February 2023

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Abstract

Purpose

The once highly anticipated antibody-based pathway-targeted therapies have not achieved promising outcomes for deadly pancreatic ductal adenocarcinoma (PDAC), mainly due to drugs’ low intrinsic anticancer activity and poor penetration across the dense physiological barrier. This study aims to develop an ultra-small-sized, EGFR/VEGF bispecific therapeutic protein to largely penetrate deep tumor tissue and effectively inhibit PDAC tumor growth in vivo.

Methods

The bispecific protein, Bi-fp50, was constructed by a typical synthetic biology method and labeled with fluorescent dyes for in vitro and in vivo imaging. Physicochemical properties, protein dual-binding affinity, and specificity of the Bi-fp50 were evaluated in several PDAC cell lines. In vitro quantitatively and qualitatively anticancer activity of Bi-fp50 was assessed by live/dead staining, MTT assay, and flow cytometry. In vivo pharmacokinetic and biodistribution were evaluated using blood biopsy samples and near-infrared fluorescence imaging. In vivo real-time tracking of Bi-fp50 in the local tumor was conducted by fibered confocal fluorescence microscopy. The subcutaneous PDAC tumor model was used to assess the in vivo antitumor effect of Bi-fp50.

Results

Bi-fp50 with an ultra-small size of 50 kDa (5 ~ 6 nm) showed an excellent binding ability to VEGF and EGFR simultaneously and had enhanced, accumulated binding capability for Bxpc3 PDAC cells compared with anti-VEGF scFv and anti-EGFR scFv alone. Additionally, bi-fp50 significantly inhibited the proliferation and growth of Bxpc3 and Aspc1 PDAC cells even under a relatively low concentration (0.3 µM). It showed synergistically enhanced therapeutic effects relative to two individual scFv and Bi-fp50x control in vitro. The half-life of blood clearance of Bi-fp50 was 4.33 ± 0.23 h. After intravenous injection, Bi-fp50 gradually penetrated the deep tumor, widely distributed throughout the whole tissue, and primarily enriched in the tumor with nearly twice the accumulation than scFv2 in the orthotopic PDAC tumor model. Furthermore, the Bi-fp50 protein could induce broad apoptosis in the whole tumor and significantly inhibited tumor growth 3 weeks after injection in vivo without other noticeable side effects.

Conclusion

The proof-of-concept study demonstrated that the ultra-small-sized, bispecific protein Bi-fp50 could be a potential tumor suppressor and an efficient, safe theranostic tool for treating PDAC tumors.

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Data Availability

The data that support the figures within this paper is available upon reasonable request.

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Acknowledgements

We thank the technical support from the Multimodal Biomedical Imaging lab, the Institute of Automation, the Chinese Academy of Sciences, and the Cold Spring Biotech Co., Ltd. In addition, we thank Beijing Gegen Biotechnology Co., Ltd. for helping prepare and purify the scFv fragments and fusion protein. This work was supported by the National Key Research and Development Program of China (No. 2017YFA0205200), the National Natural Science Foundation of Youth Program of China (No. 81901813), and the CAMS Innovation Fund for Medical Sciences (No.2021-I2M-C&T-B-067). We also thank "BioRender" for the scheme organizing. Finally, H.Y. and S.H.Y. acknowledge support from the National Institutes of Health in the U.S. (R01-CA192878).

Author information

Author notes

  1. Qian Wang and Jingyun Wang contributed equally to this work.

Authors and Affiliations

  1. Department of Diagnostic Imaging, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Acadamy of Medical Sciences and Peking Union Medical College, Beijing, 100021, People’s Republic of China

    Qian Wang & Xinming Zhao

  2. School of Pharmaceutical Sciences, Tsinghua University, Beijing, 100084, People’s Republic of China

    Jingyun Wang

  3. Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, Cambridge, MA, 02139, USA

    Hao Yan & Seok-Hyun Yun

  4. Yi-Chuang Institute of Biotechnology Industry, Beijing, 101111, People’s Republic of China

    Zheng Li

  5. CAS Key Laboratory of Molecular Imaging, Institute of Automation and Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China

    Kun Wang & Jie Tian

  6. Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, People’s Republic of China

    Hao Yan & Feiyu Kang

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  1. Qian Wang
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Contributions

Q.W., H.Y., J.T., and X.M.Z. discusses and convinced this project. H.Y. and Q.W. designed the experiments. Q.W. and Z.L. fabricated the Bi-fp50 protein. Q.W. and J.Y.W. performed the characterization, in vitro imaging, and therapy assessment. Q.W. developed the PDAC animal model. Q.W. and K.W. performed in vivo imaging experiments. Q.W. and J.Y.W. performed in vivo therapy experiments. F.Y.K. assisted in data analysis. Q. W., J.Y.W., and H.Y. prepared figures and wrote the manuscript with inputs from all co-authors.

Corresponding authors

Correspondence to Hao Yan, Jie Tian or Xinming Zhao.

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Ethics approval

The current study does not include any studies associated with human participants. All animal studies were performed according to the guidelines of the Animal Care Committee of the Chinese Academy of Medical Sciences Tumor Hospital.

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The authors declare no conflict of interest.

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This article is part of the Topical Collection on Oncology - General.

The original online version of this article was revised: The authors regret the mistakes of the assigned initial affiliations, mainly the affiliation of Hao Yan and Seok-Hyun Yun, during typesetting owing to the authors’ negligence. Furthermore, the authors apologize for the related confusion caused to readers.

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Wang, Q., Wang, J., Yan, H. et al. An ultra-small bispecific protein augments tumor penetration and treatment for pancreatic cancer. Eur J Nucl Med Mol Imaging 50, 1765–1779 (2023). https://doi.org/10.1007/s00259-023-06115-5

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