Ofatumumab monoclonal antibody affinity maturation through in silico modeling

Background: Ofatumumab, an anti-CD20 mAb, was approved in 2009 for the treatment of chronic lymphocytic leukemia. This mAb acts through immune-mediated mechanisms, in particular complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity by natural killer cells as well as antibody-dependent phagocytosis by macrophages. Apoptosis induction is another mechanism of this antibody. Computational docking is the method of predicting the conformation of an antibody-antigen from its separated elements. Validation of the designed antibodies is carried out by docking tools. Increased affinity enhances the biological action of the antibody, which in turn improves the therapeutic effects. Furthermore, the increased antibody affinity can reduce the therapeutic dose of the antibody, resulting in lower toxicity and handling cost

Methods: Considering the importance of this issue, using in silico analysis such as docking and molecular dynamics, we aimed to find the important amino acids of the Ofatumumab antibody and then replaced these amino acids with others to improve antibody-binding affinity. Finally, we examined the binding affinity of antibody variants to antigen

Results: Our findings showed that variant 3 mutations have improved the characteristics of antibody binding compared to normal Ofatumumab antibodies. Conclusion: The designed anti- CD20 antibodies showed potentiality for improved affinity in comparison to commercial Ofatumumab

Retroviral transduction of fluonanobody and the variable domain of camelid heavy-chain antibodies to chicken embryonic cells

Single domain antibodies from camel heavy chain antibodies [VHH or nanobody], are advantages due to higher solubility, stability, high homology with human antibody, lower immunogenicity and low molecular weight. These criteria make them candidates for production of engineered antibody fragments particularly in transgenic animals. To study the development of transgenic chicken using a recombinant retrovirus containing fluonanobody. The retrovirus constructs containing nanobody genes along with secretory signals and GFP gene were established and packed. The virus particle containing the obtained fusion gene was injected into the eggs in stage X. Molecular detection and protein analysis was done in the G0 chickens. The rate of hatched chicken after gene manipulation was estimated to be about 33%. Real-Time PCR assay showed that the nanobody along with GFP gene were integrated in cells of 1.2% of chickens. We conclude that although the rate of gene transfer by recombinant viruses in chickens is low, it would be possible to transfect the target camel immunoglobulin gene into chicken genome.

[Development of agglutination test for detection of isolated mannoprotein antigen from candida albicans]

Candida albicans is one of the most common causes of hospital infections. The aim of this study was to develop a rapid, easy and sensitive way to detect candida albicans. Polyclonal antibodies were prepared in rabbits. The antibodies were purified by salt concentration and Ion exchange chromatography. The purified antibody was coated onto the colloidal gold. Color change [red to blue] was observed when the purified antigens [mannoprotein of Candida albicans cell wall] were added to Polyclonal antibodies. The method was sensitive and easy. This study indicated that using colloidal gold particle agglutination method can be used for accurate and rapid candida detection Method.