Cohan, Reza Ahangari

Current treatments for choroidal neovascularization (CNV) and its associated subretinal fibrosis (SRF), major causes of vision loss, are limited by the need for frequent intravitreal injections and the emergence of drug resistance. This study evaluated the safety and efficacy of the intravitreal administration of engineered humanized anti-vascular endothelial growth factor Nanobodies, including a wild-type Nanobody (WHNb) and two mutated variants (MHNb136 and MHNb256), in a rat model of laser-induced CNV and associated SRF. Safety was assessed through in vivo electrophysiological and histopathological analyses following intravitreal injection of Nanobodies at doses of 12.5, 25, 50, and 100 µg. Efficacy was evaluated in rat models of laser-induced CNV and SRF using double immunohistochemistry for isolectin B4 and anti-collagen type I on sclerochoroidal flat mounts. Mean CNV and SRF areas in Nanobody-treated groups were compared with those in bevacizumab-treated and sham control groups. None of the Nanobodies showed retinal toxicity in safety assessments. Compared with bevacizumab, MHNb136 and MHNb256 reduced the CNV area by 1.72-fold and 1.8-fold, respectively (both p < 0.0001), whereas WHNb showed an effect nearly identical to that of bevacizumab. In addition, 12.5 µg MHNb136 and 100 µg MHNb256 reduced the SRF area by 1.3-fold (p = 0.047) and 1.6-fold (p = 0.0007), respectively, relative to bevacizumab. For CNV reduction, 12.5 µg MHNb136 was comparable to 25 µg MHNb256; both outperformed bevacizumab. For SRF reduction, 12.5 µg MHNb136 was more effective than bevacizumab and comparable to 100 µg MHNb256. These findings suggest that 12.5 µg MHNb136 may represent a cost-effective bioengineered Nanobody candidate for future clinical studies.

Background and purpose: 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, which identifies and binds to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD is an important target for developing virus-neutralizing antibodies, vaccines, and inhibitors. Experimental approach: In this study, recombinant SARS-CoV-2 RBD was expressed in E. coli BL21 (DE3) and purified and its binding activity was determined. Purification was conducted using the Ni-NTA column. ELISA. flow cytometry assays were set to evaluate the binding ability of recombinant RBD to different anti-RBD antibodies and native ACE2 receptors on HEK293A cells, respectively. Findings/Results: The SDS-PAGE analysis revealed the corresponding band at 27 kDa in the culture after induction with 0.7 mM IPTG, while the corresponding band was not observed in the culture without IPTG induction. ELISA results showed that antibodies produced in the human sera could bind to the recombinant RBD protein and the commercial anti-RBD antibody. Also, flow cytometry analysis revealed that the recombinant RBD could bind to human ACE2 on the surface of HEK293A cells. Conclusion and implication: Our outcomes displayed that the recombinant RBD expressed in the E. coli strain has biological activity and can be used as an antigen for the development of diagnosis kits and vaccines as well as a tool for screening drugs against SASR-CoV-2.