Construction and Characterization of Novel, Recombinant Immunotoxins Targeting the Her2/neu Oncogene Product: In vitro and In vivo Studies

Abstract

The goal of this study was to characterize a series of antiHer2/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/neuoverexpressing malignancies. [Cancer Res 2009;69(23):8987-95]

Suppression of hepatoma tumor growth by systemic administration of the phytotoxin gelonin driven by the survivin promoter

“…However, the autophage marker LC3-II had no changes ( Figure 6C). Subsequently, to assess whether the necrotic cell death was induced, we examined lactate LDH release, which is a marker of abrupt membrane lysis [46].The results showed that pCMV-Gel or pSur-Gel significantly increaseed the LDH release in HepG2 cells, and pCMV-Gel also significantly increased the LDH release in LO2 cells, but low in LO2 cells treated with pSurGel ( Figure 6C), which indicated that the observed cytotoxicity may be also a result of necrosis.…”

“…Although gelonin toxins have been used to treat malignant cancers many years, studies using different cell types have shown distinct mechanisms underlying the induction of cell death [45][46][47]. Also the mechanism of targeted gelonin gene therapy has no detailed reports.…”

Single-Chain Antibody-Based Immunotoxins Targeting Her2/neu: Design Optimization and Impact of Affinity on Antitumor Efficacy and Off-Target Toxicity

“…The immunotoxins were expressed in Escherichia coli strain AD494 (DE3) pLysS and purified by immobilized metal affinity chromatography (IMAC) essentially as previously described (16).…”

“…Rabbit anti-rGel antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG were used as a tracer in this assay as described previously (16).…”

“…These constructs showed highly efficient activity against Her2/neu-positive tumor cells (16). In the current study, we generated a series of rGel-containing fusion constructs composed of C6.5 and its mutants with varying affinities to Her2/neu and examined the impact of affinity on in vitro cytotoxicity, pharmacodynamics, and antitumor efficacy.…”