Selective cytotoxicity to HER2-positive tumor cells by a recombinant e23sFv-TD-tBID protein containing a furin cleavage sequence.
OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
These data suggest that recombinant e23sFv-TD-tBID has therapeutic potential for HER2-positive tumors and warrant further testing for clinical applications.
Wang
Fang
F
State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an, Shaanxi, China.
Ren
Jing
J
Qiu
Xiu-Chun
XC
Wang
Li-Feng
LF
Zhu
Qing
Q
Zhang
Ying-Qi
YQ
Huan
Yi
Y
Meng
Yan-Ling
YL
Yao
Li-Bo
LB
Chen
Si-Yi
SY
Xu
Yan-Ming
YM
Yang
An-Gang
AG
D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D049109:Cell Proliferation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D045683:Furin / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D000922:Immunotoxins / Q000276:immunology / Q000494:pharmacology*; D008279:Magnetic Resonance Imaging; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology* / Q000628:therapy*; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D051023:BH3 Interacting Domain Death Agonist Protein; D045683:Furin; D000922:Immunotoxins; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins