Design of an EGFR-targeting toxin for photochemical delivery: in vitro and in vivo selectivity and efficacy.

The number of epidermal growth factor receptor (EGFR)-targeting drugs in the development for cancer treatment is continuously increasing. Currently used EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors have specific limitations related to toxicity and development of resistance, and there is a need for alternative treatment strategies to maximize the clinical potential of EGFR as a molecular target. This study describes the design and production of a novel EGFR-targeted fusion protein, rGel/EGF, composed of the recombinant plant toxin gelonin and EGF. rGel/EGF was custom-made for administration by photochemical internalization (PCI), a clinically tested modality for cytosolic release of macromolecular therapeutics. rGel/EGF lacks efficient mechanisms for endosomal escape and is therefore minimally toxic as monotherapy. However, PCI induces selective and efficient cytosolic release of rGel/EGF in EGFR-expressing target cells by light-directed activation of photosensitizers accumulated selectively in tumor tissue. PCI of rGel/EGF was shown to be highly effective against EGFR-expressing cell lines, including head and neck squamous cell carcinoma (HNSCC) cell lines resistant to cetuximab (Erbitux). Apoptosis, necrosis and autophagy were identified as mechanisms of action following PCI of rGel/EGF in vitro. PCI of rGel/EGF was further shown as a highly tumor-specific and potent modality in vivo, with growth inhibitory effects demonstrated on A-431 squamous cell carcinoma (SCC) xenografts and reduction of tumor perfusion and necrosis induction in SCC-026 HNSCC tumors. Considering the small amount of rGel/EGF injected per animal (0.1 mg/kg), the presented in vivo results are highly promising and warrant optimization and production of rGel/EGF for further preclinical evaluation with PCI.

Design optimization and characterization of Her2/neu-targeted immunotoxins: comparative in vitro and in vivo efficacy studies.

Targeted therapeutics are potential therapeutic agents because of their selectivity and efficacy against tumors resistant to conventional therapy. The goal of this study was to determine the comparative activity of monovalent, engineered anti-Her2/neu immunotoxins fused to recombinant gelonin (rGel) to the activity of bivalent IgG-containing immunoconjugates. Utilizing Herceptin and its derived humanized single-chain antibody (single-chain fragment variable, designated 4D5), we generated bivalent chemical Herceptin/rGel conjugate, and the corresponding monovalent recombinant immunotoxins in two orientations, 4D5/rGel and rGel/4D5. All the constructs showed similar affinity to Her2/neu-overexpressing cancer cells, but significantly different antitumor activities. The rGel/4D5 orientation construct and Herceptin/rGel conjugate were superior to 4D5/rGel construct in in vitro and in vivo efficacy. The enhanced activity was attributed to improved intracellular toxin uptake into target cells and efficient downregulation of Her2/neu-related signaling pathways. The Her2/neu-targeted immunotoxins effectively targeted cells with Her2/neu expression level >1.5 × 10(5) sites per cell. Cells resistant to Herceptin or chemotherapeutic agents were not cross-resistant to rGel-based immunotoxins. Against SK-OV-3 tumor xenografts, the rGel/4D5 construct with excellent tumor penetration showed impressive tumor inhibition. Although Herceptin/rGel conjugate demonstrated comparatively longer serum half-life, the in vivo efficacy of the conjugate was similar to the rGel/4D5 fusion. These comparative studies demonstrate that the monovalent, engineered rGel/4D5 construct displayed comparable in vitro and in vivo antitumor efficacy as bivalent Herceptin/rGel conjugate. Immunotoxin orientation can significantly impact the overall functionality and performance of these agents. The recombinant rGel/4D5 construct with excellent tumor penetration and rapid blood clearance may reduce the unwanted toxicity when administrating to patients, and warrants consideration for further clinical evaluation.

Root morphology--a study of the mandibular second molar of ethnic Chinese.

The C-shaped canal system often is a challenge to optimal cleaning and shaping. This present study was aimed to determine the incidence of fused root patterns, which indicates the presence of a C-shaped canal system, in mandibular second molars of ethnic Chinese in Hong Kong. One thousand clinical and radiographic records of patients in a dental teaching hospital were randomly selected and screened. From the panoramic radiographs, 399 patients with a total of 769 mandibular second molars were suitable for analysis. Of these selected patients, 370 (92%) presented with both 37 and 47, of which 108 (29%) pairs showed the same fused root pattern. Of all 769 second molars examined, 203 (26%) presented with a radiographic Type I morphology, and 68 (9%) and 54 (7%) with Type II and III morphology, respectively. It was concluded that approximately 42% of fused-root mandibular second molars of Hong Kong Chinese patients might be associated with a C-shaped root canal system.

Different trends in modulation of NMDAR1 and NMDAR2B gene expression in cultured cortical and hippocampal neurons after lead exposure.

Exposure to heavy metal lead (Pb(2+)) has been reported to cause problems in cognitive functions of the brain, e.g. memory loss and difficulties in mental development. N-Methyl-D-aspartate receptors (NRs) are important molecules that are known to be involved in mediation of learning and memory. In order to investigate the effects of Pb(2+) on the gene expression of NR1 and NR2B subunits in neurons, primary cell cultures of rat cortical and hippocampal neurons were employed. After treatments with different concentrations of Pb(2+) ions in culture medium (0, 5, 10, 25 and 50 microM), the cellular localization of Pb(2+) in neurons was evaluated by laser scan confocal microscopy by using a Pb(2+) ion specific fluorescence probe. In addition, the gene expression of NR1 and NR2B subunits was determined by reverse transcriptase-polymerase chain reaction, immunofluorescence and Western blotting. The results of the present study showed that both cortical and hippocampal neurons accumulated intracellular Pb(2+) in accordance with the concentrations of Pb(2+) ions present in the culture medium. After Pb(2+) treatments, levels of NR1 mRNA, immunoreactivity and protein were found to be unchanged but levels of NR2B mRNA, immunoreactivity and protein were found to be significantly increased in cortical neurons. In contrast, both NR1 and NR2B mRNAs, immunoreactivity and proteins were found to be significantly decreased in hippocampal neurons. The changes in gene expression were found to be dose dependent in accordance with the Pb(2+) concentrations. The present results indicate that Pb(2+) has a differential effect on the expression of NR1 and NR2B subunits in cortical and hippocampal neurons, respectively. It is likely that the toxic effects of Pb(2+) may cause differential damage to different types of memory that are mediated by cortical and hippocampal neurons, respectively.

A novel recombinant fusion toxin targeting HER-2/NEU-over-expressing cells and containing human tumor necrosis factor.

Over-expression of the proto-oncogene HER2/neu in breast cancer and certain other tumors appears to be a central mechanism that may be partly responsible for cellular progression of the neoplastic phenotype. Transfection of mammalian cells and over-expression of HER2/neu appears to result in reduced sensitivity to the cytotoxic effects of tumor necrosis factor (TNF) and reduced sensitivity to immune effector killing. The single-chain recombinant antibody sFv23 recognizes the cell-surface domain of HER2/neu. The cDNA for this antibody was fused to the cDNA encoding human TNF, and this sFv23/TNF fusion construct was cloned into a plasmid for expression in Escherichia coli. The fusion protein was expressed and purified by ion-exchange chromatography. SDS-PAGE demonstrated a single band at the expected m.w. (43 kDa). Western analysis confirmed the presence of both the antibody component and the TNF component in the final fusion product. The fusion construct was tested for TNF activity against L-929 cells and found to have biological activity similar to that of authentic TNF (SA 420 nM). The scFv23/TNF construct bound to SKBR-3 (HER2-positive) but not to A-375 human melanoma (HER2-negative) cells. Cytotoxicity studies against log-phase human breast carcinoma cells (SKBR-3-HP) over-expressing HER2/neu demonstrate that the sFv23/TNF fusion construct was 1, 000-fold more active than free TNF. Tumor cells expressing higher levels of HER2/neu (SKBR-3-LP) were relatively resistant to both the fusion construct and native TNF. These studies suggest that fusion constructs targeting the HER2/neu surface domain and containing TNF are more effective cytotoxic agents in vitro than native TNF and may be effective against tumor cells expressing intermediate, but not high, levels of HER2/neu.

Comparative cytotoxicity and pharmacokinetics of antimelanoma immunotoxins containing either natural or recombinant gelonin.

Immunotoxins are a class of targeted therapeutic agents under development by various research groups. The murine monoclonal antibody designated ZME-018 recognizes a high molecular weight glycoprotein present on most human melanoma cells and biopsy specimens and has been utilized for clinical imaging studies in patients with melanoma. The plant toxin gelonin is a ribosome-inactivating protein (RIP) with n-glycosidase activity similar to that of ricin A chain. In previous studies by our group, the gelonin toxin was sequenced, cloned and expressed in E. coli. The purified recombinant gelonin (RG) was found to have identical protein synthesis inhibitory activity to that of natural gelonin (NG). For comparative purposes, chemical conjugates of antibody ZME and either RG or NG were produced using the heterobifunctional crosslinking reagents SPDP and SMPT. The ZME-NG and ZME-RG immunotoxins were found to be 10(4)- to 10(5)-fold more cytotoxic to antigen-positive human melanoma cells than free toxin. NG toxin alone was cytotoxic to intact cells (IC(50) = 100 nM) while RG was nontoxic to cells at doses up to 1 microM. Both ZME-NG and ZME-RG immunoconjugates were nontoxic to antigen-negative (Me-180) cells. ZME-RG immunotoxins constructed with the more stable SMPT reagent were slightly more effective in culture than conjugates made with SPDP. Tissue distribution studies in tumor-bearing nude mice demonstrated that tumor uptake of the ZME-RG immunotoxin was similar to that of the intact ZME antibody with reduced distribution to normal organs compared to an immunoconjugate produced with NG. Pharmacokinetic studies showed that the terminal-phase plasma half-life of ZME-RG was similar to that of ZME itself (42 h vs 50 h) and almost threefold higher than that of ZME-NG (11.5 h). The area under the concentration curve (Cxt) for ZME-RG was 50% lower than that for ZME due to an increased apparent volume of distribution (Vd(a)) but was almost tenfold higher than the Cxt for ZME-NG. These studies suggest that immunoconjugates comprising RG demonstrate identical in vitro cytotoxic effects to immunoconjugates produced with NG and immunotoxins with RG display improved in vivo pharmacodynamics and tissue distribution compared to immunotoxins containing NG.