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Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System
Published on: August 8, 2012
Selective cytotoxicity to HER2-positive tumor cells by a recombinant e23sFv-TD-tBID protein containing a furin
Fang Wang1, Jing Ren, Xiu-Chun Qiu
1State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an, Shaanxi, China.
Purpose:
The HER2 antigen is a recognized target on breast cancer cells for immunotherapy. To overcome the immunogenicity and systemic toxicity of traditional immunotoxins, a novel human immunoproapoptotic molecule was developed and its antitumor activity was investigated.
Experimental Design:
Recombinant e23sFv-TD-tBID, consisting of a single-chain anti-HER2 antibody fragment linked to a human active truncated Bid by a 10-amino acid residue furin cleavage sequence, was bacterially expressed. Purified e23sFv-TD-tBID was tested for binding, internalization, and cytotoxic activity in cell and for tumor localization and antitumor activity in athymic nude mice bearing established human tumor xenografts.
Results:
e23sFv-TD-tBID selectively binds to HER2-positive cells and induces apoptotic cell death in vitro and in vivo. An investigation of its mechanism of action has revealed that e23sFv-TD-tBID was internalized on binding to the surface of HER2-positive tumor cells, proteolytically cleaved and transported directly to cytosol. The antitumor activity of e23sFv-TD-tBID was shown in a dose-dependent manner when injected i.p. into immunodeficient mice bearing human breast carcinomas. Moreover, this immunoproapoptotic protein, either given as a single dose or in combination with chemotherapy agents, significantly inhibited tumor growth without any observed toxic side effects on mice. Magnetic resonance imaging further showed the specific targeting and good penetration of tumors by e23sFv-TD-tBID in vivo. The therapeutic value of e23sFv-TD-tBID to human was shown by its cytotoxic effects on primary patient-derived breast tumor cells but not on endothelial cells.
Conclusion:
These data suggest that recombinant e23sFv-TD-tBID has therapeutic potential for HER2-positive tumors and warrant further testing for clinical applications.
Insights
A novel immunoproapoptotic molecule, e23sFv-TD-tBID, targets HER2-positive breast cancer cells, inducing apoptosis with minimal toxicity. This recombinant protein shows significant antitumor activity in preclinical models, suggesting potential for clinical application.
Area of Science:
- Oncology
- Immunotherapy
- Molecular Biology
Background:
- The HER2 antigen is a validated target for breast cancer immunotherapy.
- Traditional immunotoxins face challenges with immunogenicity and systemic toxicity.
Purpose of the Study:
- To develop and investigate the antitumor activity of a novel human immunoproapoptotic molecule targeting HER2.
- To overcome limitations of existing immunotoxin therapies.
Main Methods:
- Recombinant e23sFv-TD-tBID, an anti-HER2 antibody fragment fused to truncated Bid, was engineered and expressed.
- Activity was assessed through binding, internalization, and cytotoxicity assays in vitro and in vivo models (athymic nude mice).
Main Results:
- e23sFv-TD-tBID selectively bound and induced apoptosis in HER2-positive cells.
- The molecule was internalized, cleaved, and delivered to the cytosol, mediating dose-dependent antitumor effects in mice.
- Significant tumor growth inhibition was observed with no apparent toxicity, and MRI confirmed tumor targeting and penetration.
- Cytotoxic effects were confirmed on patient-derived tumor cells but not on endothelial cells.
Conclusions:
- Recombinant e23sFv-TD-tBID demonstrates significant therapeutic potential for HER2-positive tumors.
- Further clinical investigation is warranted based on promising preclinical data.
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