A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer–small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF–TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein–protein interactions.

27 OXIDATIVE STRESS OF LIVER IN TRANSGENIC PIGLETS WITH MULTIPLE COPIES OF TRANSGENES SOLUBLE HUMAN TUMOUR NECROSIS FACTOR RECEPTOR TYPE Ig-Fc AND HUMAN HEME OXYGENASE-1

It has been demonstrated that transgene expression is associated with copy number in transgenic animals. Here in, we generated 7 genetically modified pigs expressing both soluble human tumour necrosis factor receptor type Ig-Fc (shTNFRI-Fc) and human heme oxygenase-1 (HO-1). 1 day after Caesarean section, all transgenic cloned piglets showed postnatal death. In the present study, the transgene copy number, H2O2 and superoxide dismutase (SOD) levels in cloned piglet liver were examined to identify the relationship between transgene copy number and oxidative stress of postnatal liver. In this study, 2,209 cloned embryos using somatic cells with 15 copies of shTNFRI-Fc and HO-1 were produced by somatic cell nuclear transfer, and transferred into 6 synchronized recipient sows. Among them, pregnancies were identified in 4 recipients using ultrasonography and only 1 recipient was maintained until full term. In total, 7 cloned piglets were delivered by the Caesarean section. On the next day, they showed postnatal death with clinical symptoms such as dyspnea (Group A). As control group, 292 cloned embryos produced from the cells with at least 4 copies of 2 transgenes shTNFRI-Fc and HO-1 were transferred into a synchronized recipient and pregnancy was identified. Two cloned piglets were delivered normally and maintained healthy. The liver of a live cloned piglet with at least 4 copies (Group B) at 2 days after the Caesarean section was isolated and compared with those of dead 7 cloned piglets (Group A) for HO-1, shTNFRI-Fc, H2O2, and SOD by ELISA analysis. The transgene copy number and expression of shTNFRI-Fc and HO-1 were confirmed by genomic DNA PCR, quantitative real-time PCR (qRT-PCR) and ELISA with appropriate antibodies. Statistical analysis was performed using Graphpad Prism. Level of HO-1, shTNFRI-Fc, H2O2, and SOD ELISA results of each piglets were analysed by unpaired t-test with Welch’s correction. While a transgenic piglet (Group B) had at least 4 copy numbers, all dead cloned piglets (Group A) showed 15 copy numbers. A high level of transgene HO-1 and shTNFRI-Fc expression of liver-derived cells in cloned piglets (Group A) was significantly identified compared with those of a transgenic piglet (Group B) by qRT-PCR and ELISA. While the H2O2 level in cloned piglet liver with 15 copy numbers (Group A) was significantly higher (P < 0.05), the SOD level was lower than those of a cloned pig (Group B; P < 0.05). These results demonstrated that multiple copy numbers could affect the level of oxidative stress in cloned piglet liver. It also affected the transgene expression levels and mortality of cloned piglets. This study was supported by National Research Foundation (#2015R1C1A2A01054373. 2016M3A9B6903410), Ministry of Trade, Industry & Energy (#10048948), Korea Institute of Planning and Evaluation for Technology in food, agriculture, forestry and fisheries (#114059–03–2-SB010), Research Institute for Veterinary Science, Natural Balance and the BK21 plus program.

Integration of binding peptide selection and multifunctional particles as tool-box for capture of soluble proteins in serum

In this paper, we report on a general approach for the detection of a specific tumoural biomarker directly in serum. Such detection is made possible using a protein-binding peptide selected through an improved phage display technique and then conjugated to engineered microparticles (MPs). Protein biomarkers represent an unlimited source of information for non-invasive diagnostic and prognostic tests; MP-based assays are becoming largely used in manipulation of soluble biomarkers, but their direct use in serum is hampered by the complex biomolecular environment. Our technique overcomes the current limitations as it produces a selective MP—engineered with an antifouling layer—that ‘captures’ the relevant protein staying impervious to the background. Our system succeeds in fishing-out the human tumour necrosis factor alpha directly in serum with a high selectivity degree. Our method could have great impact in soluble protein manipulation and detection for a wide variety of diagnostic applications.