Abstract
The goal of this study was to characterize a series of anti-Her2/neu immunotoxin constructs to identify how different antibodies and linker choices affect the specificity and cytotoxicity of these proteins. We constructed a series of immunotoxins containing either the human single-chain antibody (scFv) C6.5 or the murine scFv e23 fused to the highly toxic recombinant gelonin (rGel) molecule. Based on the flexible GGGGS linker (L), the fusion construct C6.5-L-rGel was compared with e23-L-rGel to evaluate the specific cytotoxic effects against Her2/neu-positive and Her2/neu-negative tumor cells. Both constructs retained the specificity of the original antibody as well as the biological activity of rGel toxin. The two constructs displayed similar cytotoxicity against different carcinoma cells. We additionally introduced the modified linkers TRHRQPRGWEQL (Fpe) and AGNRVRRSVG (Fdt), which contained furin cleavage sites, to determine the effect of these design changes on stability and cell killing efficiency. The introduction of furin cleavage linkers (Fpe or Fdt) into the molecules resulted in dissimilar sensitivity to protease cleavage compared with the constructs containing the L linker, but very similar intracellular rGel release, cytotoxic kinetics, and induction of autophagic cell death in vitro. Xenograft studies with SKOV3 ovarian tumors were done using various C6.5/rGel constructs. C6.5-L-rGel was more efficient in tumor inhibition than constructs containing furin linkers, attributing to a higher stability in vivo of the L version. Therefore, our studies suggest that human C6.5-L-rGel may be an effective novel clinical agent for therapy of patients with Her2/neu-overexpressing malignancies.
Highlights
The Her2/neu proto-oncogene encodes a 185-kDa transmembrane glycoprotein kinase with extensive homology to the epidermal growth factor receptor (HER1; refs. 1–3)
We previously reported in vitro characterization and in vivo antitumor efficacy studies of an immunotoxin composed of the human chimeric anti-Her2/neu antibody (BACH-250) chemically conjugated to recombinant gelonin. rGel is a 29-kDa ribosome-inactivating plant toxin with a potency and mechanism of action similar to that of ricin toxin A-chain, but with improved stability and reduced toxicity [18, 19]
The initial rGel-based immunotoxins consisted of a flexible L linker (GGGGS) tethering the COOH terminus of e23 or C6.5 to Characterization of e23-L-rGel and C6.5-L-rGel Immunotoxins
Summary
The Her2/neu proto-oncogene encodes a 185-kDa transmembrane glycoprotein kinase with extensive homology to the epidermal growth factor receptor (HER1; refs. 1–3). Amplification of the gene and overexpression of the Her2/neu protein product in tumor cells have been well described in numerous human cancers, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). A number of approved immunotherapeutic agents are directed at tumors that express high levels of Her2/neu, such as the monoclonal antibody trastuzumab (Herceptin), and small-molecule inhibitors such as gefitinib (Iressa) have shown promising results, but the development of resistance to treatment remains a well-known problem [7, 8]. To enhance its clinical potential, cell-surface Her2/ neu has been targeted using antibody-drug conjugates [9] or immunotoxins, composed of plant or bacterial toxins linked with a targeting molecule composed of monoclonal antibodies or antibody fragments [10, 11]. Further complications could result from nonspecific binding of foreign proteins to vascular endothelial cells, leading to vascular leak syndrome and, interstitial edema and organ failure [14, 15]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
