Treatment of hepatocellular carcinoma in a mouse xenograft model with an immunotoxin which is engineered to eliminate vascular leak syndrome

Abstract

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin and interleukin-2 therapy. It has been evidenced that VLS-inducing molecules share a three-amino acid consensus motif, (x)D(y), which may be responsible for initiating VLS. Here we have constructed a recombinant immunotoxin (SMFv-PE38KDEL) by genetically fusing PE38KDEL to a single-chain antibody derived from SM5-1 monoclonal antibody, which has a high specificity for melanoma, hepatocellular carcinoma and breast cancer. In order to eliminate VLS induced by this PE38KDEL-based immunotoxin, a panel of mutants were generated by changing amino acid residues adjacent to its three (x)D(y) motifs in the three-dimensional structure. One of the SMFv-PE38KDEL mutants, denoted as mut1, displayed a similar protein synthesis inhibitory in a reticulocyte lysate translation assay compared to the wild-type SMFv-PE38KDEL (wt). The in vitro cytotoxicity assay indicated that mut1 specifically killed SM5-1 binding protein-positive tumor cells, although its cytotoxicity was slightly less than wt. In contrast, mut1 was shown to be much weaker in inducing VLS in mice than wt. The LD(50) values of wt and mut1 in mice were investigated with the result that the LD(50) of mut1 was about tenfold higher than that of wt. The in vivo antitumor activity of wt and mut1 were also compared in tumor-bearing nude mice. Both wt and mut1 were effective in inhibiting the tumor growth but mut1 showed improved therapeutic efficacy. These studies suggest mut1 may be a novel PE-based immunotoxin with much less toxicity for clinical use.

Fig. 1

Ribbon diagram of PE38KDEL. Ribbon representation of the three-dimensional structure of PE38KDEL, with the GDL (348–350), GDV (430–432) and GDL (605–607) motifs in white, and the amino acid resides within 5Å of the three structural motifs in blue. The model was generated and optimized by the INSIGHT II program. a PE38KDEL, b GDL (348–350) motif, c GDV (430–432) motif, d GDL (605–607) motif

Fig. 2

SDS-PAGE and Western blot analysis of purified recombinant immunotoxins. a SDS-PAGE analysis of purified recombinant immunotoxins under nonreducing conditions. b SDS-PAGE analysis of purified recombinant immunotoxins under reducing conditions. c Western blot analysis of purified recombinant immunotoxins. Lane 1 molecular weight protein markersm, Lane 2 mut1, Lane 3 mut2, Lane 4 mut3, Lane 5 mut4, Lane 6 mut5, Lane 7 mut6, Lane 8 mut7, Lane 9 mut, Lane 10 wt

Fig. 3

Vascular leak syndrome in mice induced by various immunotoxins. The hydrothorax and lungs of the ICR mice were harvested 24 h after administration of 1 mg/kg (a) or 3 mg/kg (b) of different immunotoxins or PBS. The wet/dry weight ratio of hydrothorax and lungs was determined. Data are presented as the mean ± SD (n = 6). N/A indicates data are not available. Asterisks indicated a significant difference from PBS-treated mice (*, P < 0.01 by Student’s unpaired t test)

Fig. 4

Binding of FITC-SM5-1 to ch-hep-3 cells in the presence of increasing concentrations of wt or mut1. ch-hep-3 cells at 1 × 10⁶ cells/ml were incubated with sub-saturated concentration of FITC-SM5-1 and increasing concentrations of wt, mut1 or CD25-PE38 for 1 h at 4°C. The cells were then washed and analyzed by FCM. Maximal fluorescence means the mean channel fluorescence on the X-axis obtained in the absence of immunotoxin. CD25-PE38 is a recombinant immunotoxin composed of an anti-CD25 single chain antibody fused to PE38KDEL. All data were expressed as the mean of triplicate samples

Fig. 5

Antitumor effect of various immunotoxins in nude mice. Groups of six nude mice were s.c. inoculated with 5 × 10⁶ ch-hep-3 cells. Once tumors reached about 50 mm³ in size, the mice were treated with PE38KDEL (0.5 mg/kg), CD25-PE38 (0.5 mg/kg), SMFv (0.5 mg/kg), wt (0.5 mg/kg), mut1 (0.5 mg/kg), mut1(1.2 mg/kg) or mut1 (5 mg/kg) twice daily for three consecutive days. The control group received PBS only. Data are expressed as mean tumor volume ± SD (n = 6)