Immunotoxins, as a class of antitumor agents, consist of tumor-selective ligands linked to highly toxic protein molecules. This type of modified antibody has been designed for the therapy of cancers and a few viral infections. In this study, we designed immunotoxin consisting of mouse programmed cell death protein-1 (PD1), which genetically fused to diphtheria toxin (DT) subunit A (DT386). DNA construct was cloned, expressed in a bacterial system, purified, and confirmed by western blotting. The immunotoxin potency in the treatment of tumorous C57BL/6 mice was evaluated. Immunotoxin was injected intratumoral to mice, and through eight injections, 67% of the tumor volume of the test group started shrinking dramatically. On the contrary, the tumor size of the control group, treated with phosphate-buffered saline, continued its growth. The successful targeting of solid tumor cells by PD1-DT immunotoxin demonstrates the potential therapeutic utility of these conjugates.
Background: Overexpression of programmed cell death ligand 1 (PD-L1) in tumor cells and subsequent interaction with the programmed cell death protein 1 (PD-1) in tumor-infiltrating T cells cause an immune evasion of the tumor from cytotoxic T-cells. Therefore, inhibiting such interaction by a recombinant PD-1 can hinder tumor growth and extend the survival rate. Methods: The mouse extracellular domain of PD-1 (mPD-1) was expressed in E. coli BL21 (DE3) strain and purified using nickel affinity chromatography. The binding ability of the purified protein to human PD-L1 was studied using ELISA. Finally, the tumor-bearing mice were used to evaluate the potential antitumor effect. Results: The recombinant mPD-1 showed a significant binding capacity to human PD-L1 at the molecular level. The tumor size significantly decreased in the tumor-bearing mice after the intra-tumoral injections of mPD-1. Moreover, the survival rate increased significantly after eight weeks of monitoring. The histopathology revealed the necrosis in the tumor tissue of the control group compared to the mPD-1 received mice. Conclusions: Our outcomes propose that interaction blockade between PD-1 and PD-L1 is a promising approach for targeted tumor therapy.
SARS-CoV-2 causes a severe respiratory disease known as COVID-19 and is responsible for a global viral pandemic. The SARS-CoV-2 receptor binding domain (RBD) is located on the spike protein (S), which is dedicated for identifying and binding to the angiotensin converting enzyme 2 (ACE2) receptor. The RBD is an important target for development of virus neutralizing antibodies, vaccines, and inhibitors. In this study, recombinant SARS-CoV-2 RBD was expressed in E. coli BL21 (DE3) and purified as well as its binding activity was determined. Purification was conducted by Ni-NTA column. ELISA and flow cytometry assays were conducted to evaluate the binding ability of recombinant RBD to different anti-RBD antibodies and native ACE2 receptor on HEK293A cells, respectively. ELISA results showed that antibodies produced in the human sera could bind to the recombinant RBD protein as well as the commercial anti-RBD antibody. Also, flow cytometry analysis showed that the recombinant RBD was able to bind to human ACE2 on the surface of HEK293A cells. Our outcomes displayed that the recombinant RBD expressed in E. coli strain has biological activity and can be used as an antigen for development of diagnosis kits and vaccines as well as a tool for screening drugs against SASR-CoV-2.
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