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Updated: Aug 8, 2025
A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer
Published on: September 13, 2022
Targeted EpCAM-binding for the development of potent and effective anticancer proteins
Zhao Liu1, Chen Zhang1, Beiming Cui2
1Department of Biomedical Engineering, Southern University of Science and Technology (SUSTech), Shenzhen, China.
Abstract:
Protein-based cancer therapies are considered an alternative to conventional anticancer regimens, providing multifunctional properties while showing low toxicity. However, its widespread use is limited by absorption and instability issues, resulting in higher dosage requirements and a prolonged onset of bioactivity to elicit the desired response. Here, we developed a non-invasive antitumor treatment using designed ankyrin repeat protein (DARPin)-anticancer protein-conjugate that specifically targets the cancer biomarker, epithelial cell adhesion molecule (EpCAM). The DARPin-anticancer proteins bind to EpCAM-positive cancer cells and improve the in vitro anticancer efficacy by over 100-folds within 24 h, where the DARPin-tagged human lactoferrin fragment (drtHLF4) IC50 value is within the nanomolar range. Orally administered drtHLF4 was readily absorbed into the systemic flow of the HT-29 cancer murine model, exerting its anticancer effect on other tumors in the host body. Orally administered drtHFL4 cleared HT29-colorectal tumors using a single dose, whereas intratumoral injection cleared HT29-subcutaneous tumors within three doses. This approach addresses the limitations of other protein-based anticancer treatments by providing a non-invasive anticancer therapy with improved potency and tumor-specificity.
Insights
A novel protein therapy using designed ankyrin repeat protein-anticancer protein conjugates targets EpCAM-positive cancer cells. This non-invasive treatment shows enhanced efficacy and oral absorption, clearing tumors effectively.
Area of Science:
- Biotechnology
- Oncology
- Protein Engineering
Background:
- Protein-based cancer therapies offer low toxicity but face absorption and stability challenges.
- Existing protein therapies require high doses and have slow-acting bioactivity.
Purpose of the Study:
- To develop a non-invasive, highly potent, and tumor-specific protein-based anticancer therapy.
- To overcome the limitations of conventional protein therapeutics for cancer treatment.
Main Methods:
- Conjugation of designed ankyrin repeat proteins (DARPin) with an anticancer protein to target epithelial cell adhesion molecule (EpCAM).
- In vitro assessment of anticancer efficacy and IC50 values of the DARPin-anticancer protein conjugate (drtHLF4).
- In vivo evaluation of oral and intratumoral administration of drtHLF4 in a HT-29 cancer murine model.
Main Results:
- DARPin-anticancer proteins demonstrated over 100-fold in vitro anticancer efficacy against EpCAM-positive cells within 24 hours.
- The drtHLF4 conjugate exhibited nanomolar IC50 values, indicating high potency.
- Orally administered drtHLF4 was absorbed systemically and cleared colorectal tumors in mice with a single dose.
- Intratumoral injection of drtHLF4 cleared subcutaneous tumors within three doses.
Conclusions:
- Developed a non-invasive, orally available protein-based cancer therapy with improved potency and tumor specificity.
- The DARPin-anticancer protein conjugate approach effectively addresses limitations of traditional protein-based therapies.
- This strategy offers a promising alternative for cancer treatment with enhanced efficacy and reduced dosage requirements.
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