Recombinant Newcastle Disease virus Expressing IL15 Demonstrates Promising Antitumor Efficiency in Melanoma Model.

Recombinant Newcastle Disease Virus (rNDV) has shown oncolytic therapeutic effect in preclinical studies. Previous data indicate that rNDV carrying IL2 has shown promise in cancer therapy. Due to the significant side effects of IL2, IL15 has been introduced into cancer therapy. A number of studies have suggested that IL15 efficiently enhances the activities of CTL and NK cells and inhibits the tumor recurrence and metastasis. Furthermore, IL15 is less toxic than IL2. Therefore, we hypothesize that a recombinant NDV expressing IL15 would be a promising agent for the treatment of malignant tumors. The human IL15 gene or IL2 gene was incorporated into the genome of lentogenic LaSota strain at the position between the HN and L genes (namely rNDV-IL15 or rNDV-IL2). The two viruses efficiently infected tumor cells and expressed IL15 or IL2 protein. Melanoma tumor-bearing mice were treated by intra-tumoral (i.t.) injection of rNDV-IL15 or rNDV-IL2. Both rNDV-IL15 and rNDV-IL2 effectively suppressed tumor growth compared with rNDV. The 120-day survival rate of rNDV-IL15- treated group was 12.5% higher than that of rNDV-IL2 group, although the difference was not statistically significant, both recombinant viruses had strong abilities to induce CD41 T cell and CTL cell responses. However, rNDV-IL15 significantly induced more IFN-γ release and stimulated more CD81 T cells infiltration in the tumor sites compared with rNDV-IL2. In the tumor re-challenged experiment, the survival rates of rNDV-IL15 group and rNDV-IL2 group were statistically higher than that of PBS group. The survival rate of rNDV-IL15 group was 26.67% higher than that of rNDV-IL2 group although the difference was not statistically significant. In conclusion, rNDV-IL15 is a promising antitumor agent against melanoma.

[The synergistic effect of FGF-21 and insulin on regulating glucose metabolism and its mechanism].

Previous studies proposed that the synergistic effect of fibroblast growth factor-21 (FGF-21) and insulin may be due to the improvement of insulin sensitivity by FGF-21. However, there is no experimental evidence to support this. This study was designed to elucidate the mechanism of synergistic effect of FGF-21 and insulin in the regulation of glucose metabolism. The synergistic effect of FGF-21 and insulin on regulating glucose metabolism was demonstrated by investigating the glucose absorption rate by insulin resistance HepG2 cell model and the blood glucose chances in type 2 diabetic db/db mice after treatments with different concentrations of FGF-21 or/and insulin; The synergistic metabolism was revealed through detecting GLUT1 and GLUT4 transcription levels in the liver by real-time PCR method. The experimental results showed that FGF-21 and insulin have a synergistic effect on the regulation of glucose metabolism. The results of real-time PCR showed that the effective dose of FGF-21 could up-regulate the transcription level of GLUT1 in a dose-dependent manner, but had no effect on the transcription level of GLUT4. Insulin (4 u) alone could up-regulate the transcription level of GLUT4, yet had no effect on that of GLUT1. Ineffective dose 0.1 mg kg(-1) FGF-21 alone could not change the transcription level of GLUT1 or GLUT4. However, when the ineffective dose 0.1 mg x kg(-1) FGF-21 was used in combination with insulin (4 u) significantly increased the transcription levels of both GLUT1 and GLUT4, the transcription level of GLUT1 was similar to that treated with 5 time concentration of FGF-21 alone; the transcription level of GLUT4 is higher than that treated with insulin (4 u) alone. In summary, in the presence of FGF-21, insulin increases the sensitivity of FGF-21 through enhancing GLUT1 transcription. Vice versa, FGF-21 increases the sensitivity of insulin by stimulating GLUT4 transcription in the presence of insulin. FGF-21 and insulin exert a synergistic effect on glucose metabolism through mutual sensitization.

[The synergism and mechanism of action of rClone30-hDR5 in combination with TRAIL on HCC].

To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.

[Improvement of yield and purity of human fibroblast growth factor-21].

Fibroblast growth factor -21 (FGF-21) is a recently discovered metabolic regulation factor, regulating glucose and lipid metabolism and increasing insulin sensitivity. FGF-21 is expected to be a potential anti-diabetic drug. Expression of FGF-21 as inclusion bodies has advantages for high yield and purity, but the bioactivity of the protein is almost totally lost after denature and renature. That is why FGF-21 is currently expressed in soluble form. As a result, the yield is considerably low. In this study, we used SUMO vector to express SUMO-human FGF-21 (SUMO-hFGF-21) in form of inclusion body. We optimized the culture conditions to increase the yield of the bioactive human fibroblast growth factor-21. We applied the hollow fiber membrane filtration column to enrich the bacteria, wash, denature and renature inclusion bodies. After affinity and gel filtration chromatography, we examined the hypoglycemic activity of FGF-21 by the glucose uptake assay in HepG2 cells. We also detected the blood glucose concentration of type 2 diabetic db/db model mice after short or long-term treatment. The results show that the yield of ihFGF-21 was 4 times higher than that of shFGF-21. The yield was 20 mg/L for ihFGF-21 vs. 6 mg/L for shFGF-21. The purity of ihFGF-21 was above 95%, while shFGF-21 was 90%. Compared with the traditional method of extracting inclusion bodies, the production cycle was about three times shortened by application of hollow fiber membrane filtration column technology, but the bioactivity did not significantly differ. This method provides an efficient and cost-effective strategy to the pilot and industrial production of hFGF-21.

[Antitumor efficacy of the recombinant Newcastle disease virus rNDV-IL15 on melanoma models].

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.

Genetically engineered Newcastle disease virus expressing interleukin 2 is a potential drug candidate for cancer immunotherapy.

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, several clinical trials have reported that mesogenic NDV is a safe and effective agent for human cancer therapy. Interleukin 2 (IL2) is a cytokine that stimulates T cell propagation to trigger innate and adaptive immunity. IL2 has been used for cancer therapy and has achieved curative effects. In this study, a recombinant NDV LaSota strain expressing human interleukin 2 (rLaSota/IL2) was generated. The ability of rLaSota/IL2 to express human IL2 was detected in the infected tumor cells. In addition, the activity of IL2 was analyzed. The antitumor potential of rLaSota/IL2 was studied by xenograph mice carrying H22 and B16-F10 cells. Tumor-specific CD4(+) and CD8(+) T cells and MHC II were also analyzed in the two tumor-bearing models. Our study showed that rLaSota/IL2 significantly stimulated tumor-specific cytotoxic T-lymphocyte (CTL) responses and increased regulatory CD4(+) and cytotoxic CD8(+) T cells proliferation. The treatment with rLaSota/IL2 led to tumor regression in tumor-bearing mice and prolonged the survival of tumor-bearing mice. Furthermore, tumor challenging experiments demonstrated that rLaSota/IL2 invoked mice a unique capacity to remember a pathogen through the generation of memory T cells, which protect the host in the event of reinfection and form adaptive immune system. The result indicates that tumor-infiltrating CD4(+) T regulatory cells may denote the effective regression of tumors. Taken together, rLaSota/IL2 has potential for immunotherapy and oncolytic therapy of cancers and may be an ideal candidate for clinical application in future cancer therapy.

Chimeric anti-IL-17 full-length monoclonal antibody is a novel potential candidate for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease, primarily manifesting as inflammatory arthritis. It is associated with chronic inflammation of the synovial joints, mostly in the hands and feet, as well as with systemic extra-articular inflammation. The chimeric anti-interleukin (IL)-17 full-length monoclonal antibody (CMa17Aab) targets IL-17A, which is an important cytokine in the pathogenesis of RA and other inflammatory disorders. In this study, we investigated whether CMa17Aab exerts therapeutic effects in a mouse model of type II collagen-induced arthritis (CIA). Mice with CIA were subcutaneously injected with the humanized CMa17Aab antibody. The effects of treatment were assessed by estimating the arthritis severity score, the extent of histological damage and bone destruction, the autoreactive humoral and cellular immune responses and the production of cytokines. Treatment with CMa17Aab exerted beneficial effects in the mice with CIA as regards clinical and histological parameters. Compared with the controls, treatment with CMa17Aab resulted in a significant alleviation of the severity of the symptoms of arthritis, by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and downregulating the expression of IL-6, IL-8, matrix metalloproteinase (MMP)-3, IL-17, IL-1ß, tumor necrosis factor (TNF)-α, receptor activator for nuclear factor-κB ligand (RANKL) and interferon (IFN)-γ in inflamed tissues. In conclusion, our study demonstrates that treatment with CMa17Aab exerts beneficial effects in mice with CIA, by preventing joint inflammation, cartilage destruction and bone damage. These preliminary results suggest that CMa17Aab is an important regulator in RA, and that it may represent a novel therapeutic agent that may prove useful in the treatment of this disease.

[Affinity optimization of an anti-hIL17A single-chain Fv antibody through error-prone PCR technique].

AIM: To optimize antibody affinity of a humanized anti-hIL17A single-chain Fv (scFV) antibody through error-prone polymerase chain reaction (PCR) so as to develop a therapeutic humanized antibody for the treatment of rheumatoid arthritis (RA). METHODS: Variable regions of heavy chain and light chain were subjected to random mutation by error-prone PCR and a scFv library was constructed by overlapping PCR. The library was screened for improved mutants by the bacterial inner membrane display technique and flow cytometry (FCM). Real-time PCR was used to detect neutralization effects of the purified scFv antibody mutants on mRNA expressions of IL-6 and IL-8 in HeLa cells stimulated by hIL17A. RESULTS: We obtained five mutants with improved affinity. FCM revealed that the affinity of three clones was greatly enhanced as compared with the parent clone. All mutants retained binding specificity to hIL17A. Real-time PCR results showed that all five mutants could block hIL17A stimulation of HeLa cells to express IL-6 and IL-8, and the neutralization effects were positively related to mutant affinity. CONCLUSION: Error-prone PCR technique is a feasible method for antibody affinity optimization in vitro, which is able to improve the affinity and neutralization capacity of antibody on the basis of its unchanged antigen binding specificity. This study provides potential drug candidates aimed for the treatment of rheumatoid arthritis and an alternative method of optimizing antibody affinity in vitro.