Generating the Engineered Form of a Nanobody Against Placenta Growth Factor with High Antiangiogenesis Potential.

Antibody engineering is a dynamic field in antibody industry. Over 30% of novel monoclonal antibodies (mAbs) in R&D and clinical trials are engineered forms. Affinity enhancement contributes to the development of new binders that are not only effective in low dose and cost but also improve some drawbacks of antibody production. After previous successful work on in silico affinity maturation of nanobody against placenta growth factor and finding the best engineered nanobodies (Mut2:S31D and Mut4:R45E), according to bioinformatic parameters and molecular dynamics (MD) simulation results, in this study we focused on experimental confirmation of affinity enhancement of a mutant form of nanobody. So, we cloned and expressed two of four mutant forms in pHEN6c vector. Affinity binding was assayed by enzyme-linked immunosorbent assay on purified mutants, with results showing that 10-time enhancement in affinity compared with the native form associated with MD simulation results. We checked the effectiveness of these mutant nanobodies in angiogenesis inhibition by human umbilical vein endothelial cell proliferation and 3D capillary tube formation. EC50 of mut2, mut4, and native in proliferation assay was 110, 140, and 190 ng/mL, respectively, and that in 3D capillary tube formation was 80, 83, and 100 ng/mL. The results of functional studies revealed strong effectiveness of Mut2 followed by Mut4 compared with the native form. Our study confirmed that in silico approach could facilitate development of novel versions of mAb with better characteristics, which could save cost and time.

Histologic effects of demineralized bone matrix on regeneration of alveolar socket in diabetic rats.

INTRODUCTION: The aim of this in vivo study was to determine the effect of demineralized bone matrix (DBM) on alveolar bone repair in type I diabetic rats. MATERIALS AND METHODS: This study was carried out on 40 adult (8 weeks-old) albino rats with an average weight of 200-250 grams. The animals were divided into four groups (n=10) as follows: group 1 nondiabetic rats, group 2, 3 and 4 were diabetic rats, group 4 rats took one unit of insulin daily. Diabetes was induced by Alloxan Monohydrate through the tail veins of the rats in groups 2-4. Only group 4 received insulin NPH 1 unit daily. After 10 days, the upper right incisors of all samples were extracted and the socket was filled with DBM in groups 3 and 4. The animals were sacrificed at the end of week 1 and 2. The specimens were prepared and stained with H&E. RESULTS: Histological results of group 4 displayed osteoblastic activity and bone formation with collagen fibers at the end of the first week and thick bone trabeculae formation in vicinity of DBM at the end of second week. In group 3, DBM showed some osteoinductivity at the end of the first week, but in some regions DBM particles were degraded by osteoclastic activity. Bone trabeculae formed with a dispersed and separate pattern at the end of second week. In group 2 hematoma and inflammation were dominant histological features at the end of first and second weeks; poor bone formation was detected in these two groups (2 and 3). In group 1, the results were as expected. CONCLUSION: It seems demineralized bone matrix simulate osteoblastic activity.