Yingqi Zhang

Myostatin dna vaccine increases skeletal muscle mass and endurance in mice

Myostatin is a transforming growth factor‐β family member that acts as a negative regulator of skeletal muscle growth. In mice, genetic disruption of the myostatin gene leads to a marked increase in body weight and muscle mass. Similarly, pharmacological interference with myostatin in vivo in mdx knockout mice results in a functional improvement of the dystrophic phenotype. Consequently, myostatin is an important therapeutic target for treatment of diseases associated with muscle wasting. To construct a therapeutic DNA vaccine against myostatin, we coupled the foreign, immunodominant T‐helper epitope of tetanus toxin to the N terminus of myostatin, and BALB/c mice were immunized with the recombinant vector. Sera from vaccinated mice showed the presence of specific antibodies against the recombinant protein. In addition, body weight, muscle mass, and grip endurance of vaccinated mice were significantly increased. Our study provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting. Muscle Nerve, 2007

Anti-tumor efficacy and pre-clinical immunogenicity of IFNα2a-NGR.

Previously studies have shown that tumor-homing peptide NGR enhances the therapeutic efficacy of human interferon α2a (IFNα2a) against tumors. Here we investigated in vivo anti-tumor effect of recombinant human IFNα2a-NGR (rhIFNα2a-NGR) against human lung adenocarcinoma cell line SPC-A-1, A549 and murine Lewis lung carcinoma (LLC) subcutaneously xenografted tumors and further assessed the immunogenicity of rhIFNα2a-NGR in Sprague Dawley (SD) rats and rhesus monkeys. We found that rhIFNα2a-NGR significantly inhibited the growth of SPC-A-1, A549 and LLC cells-xenografted tumors in a dose-dependent manner. Although the antibodies to rhIFNα were detected in the serum of SD rats and rhesus monkeys treated with rhIFNα2a-NGR, these antibodies did not cause obvious pathological consequence. Taken together, these data demonstrate that rhIFNα2a-NGR has obvious anti-tumor efficacy in vivo, perhaps due to the tumor-homing peptide NGR. Thus rhIFNα2a-NGR represents a promising novel drug for effective treatment of cancer.

A mutated human tumor necrosis factor-alpha improves the therapeutic index in vitro and in vivo.

BACKGROUNDnTumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that has cytotoxic, cytostatic and immunomodulatory effects on malignant tumors. However, clinical trials have revealed high systemic toxicity and this has hampered its utilization as an anti-cancer agent. In this study, a human TNF-alpha mutant was created and tested for its anti-tumor effects.nnnMETHODSnThe TNF mutant (recombinant mutated human TNF; rmhTNF) was prepared by protein engineering in which amino acids Pro, Ser and Asp at positions 8, 9 and 10 of TNF-alpha were substituted by Arg, Lys and Arg, and C terminal Leu157 was substituted by Phe, along with deletion of the first seven N-terminal amino acids. Prokaryotic expression recombinant vector pBV-mhTNF containing the PLPR promotor was constructed and transformed into E. coli DH5alpha. The rmhTNF was expressed in a partially soluble form in DH5alpha, purified from the supernatant of cell lysate by ammonia sulfate precipitation and two sequential chromatographic steps.nnnRESULTSnThe purified rmhTNF was >95% pure by SDS-PAGE stained with silver and high-pressure size exclusion chromatography (SEC-HPLC). Its yield was about 1.22 mg/g wet cell paste. The mutant rmhTNF exhibited an approximately 50-fold increase in cytotoxicity relative to the wild-type rhTNF on the mouse fibroblast cell line L929 in a standard cytotoxicity test, and at least and at least 50 times higher LD50 as wild type rhTNF in mice. In vivo biological activity studies carried out on tumor cell transplanted mice and nude mice also showed a more effective cytotoxicity of rmhTNF than rhTNF.nnnDISCUSSIONnThese results suggest that rmhTNF has potential for developing an effective anti-tumor reagent for some tumors.

Expression, refolding, and characterization of GFE peptide-fused human interferon-α2a in Escherichia coli

Interferon-α2a (IFN-α2a) has been used for the treatment of various viral infections and cancers for many years. However some untolerable side effects have limited its application in some aspects. To evaluate whether or not an oligopeptide containing GFE motif can home human IFN-α2a to specific tissues, a fusion gene was constructed by fusing the coding sequence of GFE peptide (CGFECVRQCPERC), which was screened from phage display peptide library, to the 3′ end of human IFN-α2a gene by recombinant DNA technique. Fusion protein rhIFN-α2a-GFE was expressed in Escherichia coli as inclusion bodies using a T7 RNA polymerase expression system, pET-22b, refolded through dialysis and purified to homogeneity to >95% of purity by affinity chromatography. Characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting demonstrated the authenticity of the fusion protein. Purified rhIFN-α2a-GFE was found to be functionally active in terms of its antiviral activity for about 2.5×108 IU/mg in vitro. Yields of the purified fusion protein were about 200 mg/L of culture medium. Tissue distribution assay in mouse showed that at 30 min IFN-α2a could be enriched sevenfold higher in lung in the targeted IFN group of mice than in the standard IFN group of mice, and last for a long time. At 1 h, IFN-α2a in the targeted IFN group was still 4.02-fold higher than that in the standard group. This confirmed that GFE peptide has the ability to selectively deliver its fusion partner IFN-α2a to lungs. The results also showed that the IFN-α2a-GFE could be specifically enriched in kidney and liver. Its distribution in kidney was concordant with the finding of GFE receptor, MDP, in kidney. However, the IFN-α2a-GFE in liver may imply some significance in pharmacology and toxicology.

Construction of a new tumour necrosis factor fusion‐protein expression vector for high‐level expression of heterologous genes in Escherichia coli

We report the construction and application of a new fusion‐protein expression plasmid (TNFHis) for Escherichia coli . The plasmid contains both PR and PL promoters and is optimized to allow a higher level of expression of mature coding sequences. It also contains a six‐histidine tag for convenient purification as well as thrombin and hydroxylamine recognition sites for cleaving heterologous protein. The potential use of this expression vector is demonstrated by comparing the expression levels of human tumour necrosis factor (TNF), interferon, interleukin 11, colony‐forming factor, osteoprotegrin and interleukin 2 in E. coli . Furthermore, all expressed TNF fusion proteins can be detected by anti‐TNFα antibody or by specific antibodies and purified by Ni2+‐nitrilotriacetate beads. The expressed TNF fusion proteins can be cleaved by hydroxylamine.

Comparative pharmacokinetics of a tumour-targeting therapy candidate rh-IFNα2a-NGR with rh-IFNα2a administered intravenously in mice and rats.

rh‐IFNα2a‐NGR is a promising anti‐tumor candidate. The aim of present study was to compare pharmacokinetics of rh‐IFNα2a‐NGR with rh‐IFNα2a.