Jingzhuang Zhao

Synergistic effects of interleukin-1β and interleukin-17A antibodies on collagen-induced arthritis mouse model.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Both interleukin-1β (IL-1β) and Interleukin-17 (IL-17) are important proinflammatory cytokines involved in the pathogenesis of RA. We investigated whether combination therapy with IL-1β and IL-17A antibodies would generate the potential for synergistic effects on a collagen-induced arthritis (CIA) mouse model. Mice with CIA were subcutaneously injected with humanized IL-1β antibody, IL-17A antibody, or combination treatment. The effects of treatment were determined by arthritis severity score, histological damage and bone destruction, autoreactive humoral and cellular immune responses and cytokine production. Treatment with IL-1β antibody or IL-17A antibody alone resulted in beneficial effects on clinical and histological parameters of CIA mice. Compared with the single antibody treatments, the combination therapy resulted in a more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-17A, IFN-γ, RANKL and MMP-3 in inflammatory tissue. In conclusion, combination treatment with humanized IL-1β and IL-17A antibodies demonstrates synergistic beneficial effects for preventing joint inflammation and cartilage destruction and bone damage in CIA mice model. These studies also provide evidence that combination with IL-1β and IL-17A antibodies may lead to a new combinatorial therapy for RA patients.

scFv antibodies against infectious bursal disease virus isolated from a combinatorial antibody library by flow cytometry.

Infectious bursal disease is an economically important disease that affects chickens worldwide. Here, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry against VP2 protein through a bacteria display technology, resulting in the enrichment of scFv. Three scFv clones with different fluorescence intensity were obtained by random colony pick up. The isolated scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis. The potential use of the selected IBDV-specific scFv antibodies was demonstrated by the successful application of the isolated antibodies in western blotting assay and ELISA.

Enhancement of the Pharmacological Efficacy of FGF-21 by Genetic Modification and PEGylation

FGF-21 is a potential candidate for the treatment of type 2 diabetes mellitus. However, the clinical application of wild-type human FGF-21 is challenging due to some limitations, such as its poor hypoglycemic potency and short in vivo half-life. In this paper, we have produced an FGF-21 mutant (ahmFGF-21) by exchanging the functional domain of hFGF-21 with that of mFGF-21 to improve the potency of FGF-21. Results showed that the ahmFGF-21 protein was more potent than wild-type hFGF-21 in stimulating glucose uptake in vitro and lowering blood glucose levels of diabetic animals. To decrease its immunogenicity and increase its biostability, the N-terminus of ahmFGF-21 was modified in a sitespecific manner with 20 kDa mPEG-propionaldehyde (mPEG-ALD). We found that the preservation time of ahmFGF-21 in vitro was significantly prolonged after PEGylation. The serum antibody levels against ahmFGF-21 in immunized rabbits with the PEGylated ahmFGF-21 were significantly reduced than those with the unmodified ahmFGF-21, and the target protein concentration in the rabbits administrated with the PEGylated ahmFGF-21 increased 9.5-fold higher than that of the unmodified ahmFGF-21. The animal experimental results showed that PEGylation of ahmFGF-21 enhanced the hypoglycemic effect in diabetic mice. These results suggest that the in vitro and in vivo hypoglycemic effects of FGF-21 are significantly enhanced by genetic modification and the metabolic pharmacology of FGF-21 in type 2 diabetic mice is improved by PEGylation at a specific site.

Bi-specific antibodies with high antigen-binding affinity identified by flow cytometry.

Using conventional approaches, the antigen-binding affinity of a novel format of bi-specific antibody (BsAb) cannot be determined until purified BsAb is obtained. Here, we show that new lipoprotein A (NlpA)-based bacteria display technology, combined with flow cytometry (FCM), can be used to detect antigen-binding affinity of BsAbs, in the absence of expression and purification work. Two formats of BsAb, scFv2-CH/CL and Diabody-CH/CL, specific for human interleukin 1β (hIL-1β) and human interleukin 17A (hIL-17A), were constructed and displayed in Escherichia coli using NlpA-based bacteria display technology. Conversion of these cells to spheroplasts, and their incubation with fluorescently conjugated antigens resulted in the selective labeling of spheroplasts expressing BsAb; enabling their antigen-binding affinity to be analyzed with FCM. The association and dissociation of BsAbs for binding to hIL-1β and hIL-17A were analyzed using FCM-based assays. The results showed that antigen-binding affinity of Diabody-CH/CL was significantly higher than that of scFv2-CH/CL. To confirm these results of FCM-based assays, BsAbs were expressed, purified and subjected to relative affinity measurements, in vitro and in vivo bioactivity analysis. The results showed that Diabody-CH/CL had greater relative affinities for both antigens, resulting in better blocking bioactivities on cellular level and effects on alleviating joint inflammation, and cartilage destruction and bone damage in collagen induced arthritis (CIA) mice model. These results indicate that BsAbs with good antigen-binding affinity can be identified by FCM-based assays without expression and purification work, and the indentified BsAb can serve as a lead compound for further drug development.

Neutralizing scFv Antibodies against Infectious Bursal Disease Virus Isolated From a Nlpa-Based Bacterial Display Library

Infectious bursal disease (IBD) considered as one of the major viral diseases threatening the poultry industry worldwide. The causative agent of the IBD is Infectious bursal disease virus (IBDV) which replicates in developing B lymphocytes in the bursa of Fabricius leading to its destruction and bursal infl ammation. In this study, we investigated a technology to produce therapeutic recombinant antibodies against IBDV in bacteria by constructing a bacterial displayed recombinant scFv library from immunized chickens, followed by screening the scFv library by fl uorescence activated cell sorting (FACS) with FITC-labeled VP2. Twelve VP2-binding scFv clones with unique sequences were obtained, with overall amino acid homology of 81.53%. The complementarity determining region (CDR) 3 in the heavy chain displayed the lowest homology, while the amino acid sequences in framework regions and CDR2 of both chains and CDR1 of the heavy chain are relatively conserved. Twelve VP2binding scFv clones were expressed in E.coli and purifi ed through denaturation and denaturation of inclusion bodies. Our ELISA results showed that all scFvs exhibited binding ability and specifi city to VP2 and various IBDV strains. In addition, two scFvs showed signifi cant neutralizing activity to IBDV (B-87 strain) as these scFvs inhibited cytopathic effect of chicken embryo fi broblast (DF1) caused by IBDV. In conclusion, our study provides a lead candidate for further development of therapeutic antibodies for IBDV infection. Research Article Neutralizing scFv Antibodies against Infectious Bursal Disease Virus Isolated from a Nlpa-Based Bacterial Display Library Tianhe Li1*, Bing Zhou2*, Tingqiao Yu3, Ning Li4, Xiaochen Guo2, Tianyuan Zhang2, Jingzhuang Zhao2, Liming Xu2, Siming Li2, Lei Ma2, Tingting Li 2, Liangjun Ding2, Mingzhe Sun2, Deshan Li2# and Jiechao Yin2# 1Beijing Obstetrics and Gynecology Hospital, Capital Medical University Beijing Maternal and Child Health Care Hospital, China 2Biopharmaceutical Lab, College of Life Science, Northeast Agricultural University, China 3College of Biological Sciences and Biotechnology, Beijing Forestry University, China 4Patent Examination Cooperation Center of the Patent Offi ce, SIPO, Beijing, China #Authors contributed equally to this study *Address for Correspondence: Tianhe Li, Beijing Obstetrics and Gynecology Hospital, Capital Medical University Beijing Maternal and Child Health Care Hospital, China, Tel: 045155190645; Email: deshanli@163.com Bing Zhou, Biopharmaceutical Lab, College of Life Science, Northeast Agricultural University, China, Tel: 0451-55190645 Submitted: 20 January 2017 Approved: 15 February 2017 Published: 21 February 2017 Copyright:

The Effect of FGF-21 on Glucose Metabolism in The Resistin-induced Insulin Resistance Liver Cells

Resistin was a cytokine associated with insulin resistance.The objective of the study is to investigate the impact of fibroblast growth factor(FGF)-21 on glucose metabolism in resistin-induced insulin resistant liver cells.A stable HepG2 cell line expressing human resistin was developed.The cell line was treated with different concentrations of insulin or FGF-21.The glucose uptake of the treated cells was measured by the method of GOD-POD,and the mRNA expression of GLUT1 and PPAR-γ of the FGF-21-treated cells were examined by real-time PCR.We successfully developed the HepG2 line which stably expressed human resistin.The results of glucose uptake showed that the sensitivity of the cell line to the insulin treatment significantly decreased,but FGF-21 could effectively stimulate the glucose uptake by the same cell line.The mRNA expression of GLUT1 was elevated in the cell line,and the GLUT1 mRNA level of the cells significantly increased after treatment by FGF-21.The expression of PPAR-γ had no significant change.The cell line in which resistin was highly expressed has a reduced susceptibility to insulin.Although the high level of resistin makes cells insulin resistance,FGF-21 can regulate the glycometabolism effectively in the cells.