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.

Treatment of hepatocellular carcinoma in mice with PE38KDEL type I mutant-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with humanized SM5-1 F(ab′) fragments

“…1-Ethyl-3-3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide were obtained from Sigma. Mut-I, PE38KDEL-I, and CD25-PE38KDEL (a control immunotoxin) were obtained as described previously (3,16). Humanized SM5-1 mAb and humanized anti-CD25 mAb were kindly provided by Shanghai Center for Cell Engineering and Antibody.…”

“…The cells were then exposed to a series of concentrations of nanoparticles or Mut-I for 2 days. Cell viability was measured using Cell Titer 96 nonradioactive cell proliferation assay kit (Promega) as described previously (3). Briefly, 20 AL MTS/PMS solution was added to each well.…”

“…SM5-1 binds to a membrane protein of about 230 kDa, which is specifically expressed on melanoma, hepatocellular carcinoma, and breast cancer cells, making it a promising candidate for cancer-targeted therapy (3)(4)(5). Previous studies showed that Mut-I had reduced toxicity and improved therapeutic efficacy than SMFv-PE38KDEL wild-type did (3). However, the problems associated with the immune response to the PE component of Mut-I still need to be resolved.…”

“…We reported previously the development of SMFv-PE38KDEL type I mutant (PE38KDEL-I; Mut-I), an immunotoxin in which a single-chain antibody (scFv) derived from mouse SM5-1 monoclonal antibody (mAb) is genetically fused to PE38KDEL-I (3). SM5-1 binds to a membrane protein of about 230 kDa, which is specifically expressed on melanoma, hepatocellular carcinoma, and breast cancer cells, making it a promising candidate for cancer-targeted therapy (3)(4)(5). Previous studies showed that Mut-I had reduced toxicity and improved therapeutic efficacy than SMFv-PE38KDEL wild-type did (3).…”

Diphtheria toxin/human B-Cell activating factor fusion protein kills human acute lymphoblastic leukemia BALL-1 cells: An experimental study

“…In 1990s, the recombinant immunotoxin technology emerged with the development of molecular biology techniques [17][18][19] BAFF is a type II transmembrane protein with N terminal intracellular domain, absent signal peptide, and C terminal extracellular domain which is highly homologous with TNF-and APOL-related leukocyte expressed ligand 2 (APRIL) of the TNF superfamily . In this technology, the encoding gene of a toxin and the ligand gene of a specific tumor cell surface molecule are fused into a recombinant plasmid.…”