ABSTRACT
During the past two decades, tumor therapy based on monoclonal antibody has been found as a confident therapeutic approach in solid tumors and hematologic malignancies. Nanobodies are the smallest fragment of an antigen-binding domain in heavy chain-only antibody originated from the Camelidae family. Accordingly, they are being recently developed rapidly as diagnostic and therapeutic agents. In this regard, targeting of angiogenic factors like Placenta growth factor (PLGF) via nanobodies show a high effectiveness. In the current study, we developed a recombinant anti-PLGF bivalent nanobody based on the affinity enhancement mutant form of anti-PLGF nanobody to suppress the angiogenesis progression. Thereafter, the bivalent nanobody (bi-Nb) was cloned and then expressed into a bacterial system. Afterward, the purity was authorized using western blot assay and the affinity was assessed using ELISA. In this regard, proliferation, 3D capillary tube formation, and migration assays were employed as functional assays. The obtained data were analyzed using t-test and P < 0.05 was considered as statistically significant. The results indicate that the bivalent nanobody could inhibit proliferation, mobility, and formation of endothelial cell capillary-like structure. Moreover, the EC50 was estimated for endothelial cell's proliferation and capillary tube's formation to be about 100 ng/ml and 65 ng/ml, respectively. Migration of MCF-7 was inhibited as about 69%, rather than the control. Accumulation of data have shown that targeting of angiogenic factors like VEGF via monoclonal antibodies or nanobodies can be useful in the suppression of tumor progression. Also, the inhibition of PLGF with monoclonal antibody indicated that it is significant in angiogenesis suppression. However, due to intrinsic properties of nanobodies, they are suggested to be used. Since the small size is rapidly removed through liver or kidney system, so it is important to use bivalent or polymeric forms for extending the half-life. Our findings indicated that the inhibition of PLGF can prevent growth and proliferation of endothelial cells and tumor cells through the bivalent nanobody. So, it is suggested as a novel therapeutic agent for angiogenesis suppression.
Subject(s)
Antibodies, Bispecific/genetics , Placenta Growth Factor/antagonists & inhibitors , Recombinant Proteins/pharmacology , Single-Domain Antibodies/genetics , Angiogenesis Inhibitors/pharmacology , Antibodies, Bispecific/metabolism , Antibodies, Bispecific/pharmacology , Antibody Affinity , Cell Movement/drug effects , Cell Proliferation/drug effects , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/growth & development , Human Umbilical Vein Endothelial Cells , Humans , MCF-7 Cells , Mutation , Placenta Growth Factor/genetics , Single-Domain Antibodies/metabolism , Single-Domain Antibodies/pharmacologyABSTRACT
Immunological disorders are among the main causes of recurrent spontaneous abortions (RSAs). Mesenchymal stem cells (MSCs) have been shown to modulate various aspects of immune responses. It seems that MSCs may improve the immunological conditions in immune-mediated RSA. The aim of this study is the reduction of resorption in RSA mouse model through MSCs therapy. The adipose-derived MSCs were administered intraperitoneal to pregnant CBA/J mice on day 4.5 of gestation in abortion-prone matting. On day 13.5 of pregnancy, abortion rates were calculated and transforming growth factor-ß (TGF-ß), interleukin 10 (IL-10), interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α) gene expression in deciduas were evaluated by real-time polymerase chain reaction (PCR). The level of TGF-ß in serum was also determined by enzyme linked immunosorbent assay (ELISA) method. The obtained results showed that MSCs therapy could reduce the abortion rate significantly in test group compared to controls. MSCs therapy also caused a significant upregulation of TGF-ß and IL-10 and downregulation of IFN-γ and TNF-α genes expression in deciduas. However, the levels of TGF-ß didn't change in mice sera. Due to the significant decrease in abortion rate, we concluded that MSCs therapy could modulate the immune responses in fetomaternal interface and protect fetus from undesirable immune responses. So, these cells might be considered as a new therapeutic for spontaneous pregnancy loss. The local upregulation of TGF-ß and IL-10 and downregulation of IFN-γ and TNF-α gene expression in decidua could be considered as one possible mechanism of immune regulation, which could protect the fetus.