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Affitoxin--a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors.

ABSTRACT: Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25% to 30% of breast cancers and has been associated with an unfavorable prognosis. Here we report the construction, purification, and characterization of Affitoxin-a novel class of HER2-specific cytotoxic molecules combining HER2-specific Affibody molecule as a targeting moiety and PE38KDEL, which is a truncated version of Pseudomonas exotoxin A, as a cell killing agent. It is highly soluble and does not require additional refolding, oxidation, or reduction steps during its purification. Using surface plasmon resonance technology and competitive binding assays, we have shown that Affitoxin binds specifically to HER2 with nanomolar affinity. We have also observed a high correlation between HER2 expression and retention of Affitoxin bound to the cell surface. Affitoxin binding and internalization is followed by Pseudomonas exotoxin A activity domain-mediated ADP-ribosylation of translation elongation factor 2 and, consequently, inhibition of protein synthesis as shown by protein expression analysis of HER2-positive cells treated with Affitoxin. Measured IC50 value for HER2-negative cells MDA-MB468 (65+/-2.63 pM) was more than 20 times higher than the value for low HER2 level-expressing MCF7 cells (2.56+/-0.1 pM), and almost 3 orders of magnitude higher for its HER2-overexpressing derivative MCF7/HER2 (62.7+/-5.9 fM). These studies suggest that Affitoxin is an attractive PE38-based candidate for treatment of HER2-positive tumors.

SUBMITTER: Zielinski R

PROVIDER: S-EPMC3402039 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Affitoxin--a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors.

Zielinski Rafal R Lyakhov Ilya I Jacobs Amy A Chertov Oleg O Kramer-Marek Gabriela G Francella Nicholas N Stephen Andrew A Fisher Robert R Blumenthal Robert R Capala Jacek J

Journal of immunotherapy (Hagerstown, Md. : 1997) 20091001 8

Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25% to 30% of breast cancers and has been associated with an unfavorable prognosis. Here we report the construction, purification, and characterization of Affitoxin-a novel class of HER2-specific cytotoxic molecules combining HER2-specific Affibody molecule as a targeting moiety and PE38KDEL, which is a truncated version of Pseudomonas exotoxin A, as a cell killing agent. It is highly soluble and does not require a ...[more]

PMID: 19752752

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Project description:We previously reported the formulation and physical properties of HER2 (human epidermal growth factor receptor 2)-specific affibody (ZHER2:342-Cys) conjugated thermosensitive liposomes (HER2(+)affisomes). Here we examined localized delivery potential of these affisomes by monitoring cellular interactions, intracellular uptake, and hyperthermia-induced effects on drug delivery. We modified ZHER2:342-Cys by introducing a glycine-serine spacer before the C-terminus cysteine (called ZHER2-GS-Cys) to achieve accessibility to cell surface expressed HER2. This modification did not affect HER2-specific binding and ZHER2-GS-Cys retained its ability to conjugate to the liposomes containing dipalmitoyl phosphatidyl choline: DSPE-PEG2000-Malemide, 96:04 mole ratios (HER2(+)affisomes). HER2(+)affisomes were either (i) fluorescently labeled with rhodamine-PE and calcein or (ii) loaded with an anticancer drug doxorubicin (DOX). Fluorescently labeled HER2(+) affisomes showed at least 10-fold increase in binding to HER2(+) cells (SK-BR-3) when compared to HER2(-) cells (MDA-MB-468) at 37°C. A competition experiment using free ZHER2-GS-Cys blocked HER2(+) affisome-SK-BR-3 cell associations. Imaging with confocal microscopy showed that HER2(+) affisomes accumulated in the cytosol of SK-BR-3 cells at 37°C. Hyperthermia-induced intracellular release experiments showed that the treatment of HER2(+) affisome/SK-BR-3 cell complexes with a 45°C (±1°C) pre-equilibrated buffer resulted in cytosolic delivery of calcein. Substantial calcein release was observed within 20min at 45°C, with no effect on cell viability under these conditions. Similarly, DOX-loaded HER2(+)affisomes showed at least 2- to 3-fold higher accumulation of DOX in SK-BR-3 cells as compared to control liposomes. DOX-mediated cytotoxicity was more pronounced in SK-BR-3 cells especially at lower doses of HER2(+)affisomes. Brief exposure of liposome-cell complexes at 45°C prior to the onset of incubations for cell killing assays resulted in enhanced cytotoxicity for affisomes and control liposomes. However, Doxil (a commercially available liposome formulation) showed significantly lower toxicity under identical conditions. Therefore, our data demonstrate that HER2(+)affisomes encompass both targeting and triggering potential and hence may prove to be viable nanodrug delivery carriers for breast cancer treatment.

Dataset Information

Affitoxin--a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors.

Publications

Affitoxin--a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors.

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