Dec - 205 Targeted Antibody Preparation And Humanized Reformation

Monoclonal antibody has great application perspective as biomedical therapeutics. It can be used as modulator of the immune system in many immune disorder disease, as well as vector to target medicine to specific sites. DEC-205as pattern recognition receptor (PRR) expressed on Dentritic cells (DC), can capture antigens specifically, and present it to immune effector cells after processing. Anti-DEC-205antibody can greatly increase the antigen presentation by DCs. In vitro experiment showed that once linked to anti-DEC-205antibody, the antigen could be presented by more than1000times. Thus, anti-DEC-205antibody could be used as vaccine vector for many diseases, such as chronic virus infection and tumors.We generated an anti-DEC-205monoclonal antibody1-17-2by immunization of rat and cell fusion technology. The antibody was identified as IgG1and Kappa isotype respectively. It could recognize DEC-205molecules on human DCs and induce internalization. Through ImageStream experiment, we found DC could intensively internalize1-17-2antibody after binding.1-17-2antibody can be used as antigen targeting vector in disease to increase the antigen presentation. However, it must be humanized to be used in human body because of its rat origination.There are many methods designed for antibody humanization, which could be sumerized into two categories:approaches based on rational design and approaches based on empirical. CDR graft, which belongs to the first category, is the most fundamental method. The principle of this method is to graft CDR (complementarity determining regions) of parent antibody to a human IgG FR (frame region) template, in order to largely substitute the non-human amino acids and keeps its binding affinity meanwhile. However, the affinity usually decrease or lost completely after grafting. The two key steps in CDR graft are the chose of FR template and finding the key residues that need to be mutated back. Both of the steps could impose great impact on the final affinity, which add the difficulty to the antibody humanization.In this article, we developed a novel antibody humanization method based on epitope scanning and MD (molecular dynamics) simulation in order to humanized1-17-2antibody. First, we used homology modeling and MD simulation to build the precise model of1-17-2Fab. A novel epitopes scanning algorithm was designed to identify antigenic residues in the framework regions (FRs) which need to be mutated to human counterpart in the humanization process. In1-17-2, we identified30residues to be substituted. However, after30substitutions, the CDR conformation had been altered, which may compromise its affinity. Next, we used virtual mutation and MD simulation to assess the conformational impact imposed by all the30mutations. By comparing the root-mean-square deviations (RMSDs) of CDRs, we found five residues defined as key residues, whose mutations would destroy the original conformation of CDRs. They need to be back mutated to rescue the antibody binding affinity.Next, we conducted experiment to validate our prediction on the key residues. We constructed expression vectors of chimera antibody, humanized antibody and refined antibody respectively as designed. The expression vectors were used to produce antibodies in vitro. We used surface plasmon resonance (SPR) assay to measure binding kinetics characters of those antibodies. It showed that humanized antibody has a reduced binding affinity compared with chimera antibody, and the binding affinity of refined antibody has been rescued to the level of original chimera antibody. The experiment result confirmed our prediction that through back mutation on key residues, humanized antibody could restore its affinity. Thus, we successfully make a humanized version of antibody1-17-2without lost of binding affinigy.Through detect assay by confocal and FCM, we confirmed that the refined antibody could induce internalization on DC as well as the original antibody1-17-2. Thus, the humanized anti-human DEC-205antibody has potential value to be used as a vector for therapeutic vaccine.Lastly, we used molecular docking to predict the binding pose by1-17-2Fab and DEC-S. Through analysis of docking surface within the binding pose, we made a prediction on the epitope of DEC-S and also the SDR of the1-17-2Fab. Those predictions could provide key information in the future improvement of the therapeutic antibody.In summary, we generated an anti-hman DEC-205monoclonal antibody, which could induce internalization on DCs, and then successfully made it humanized while retaining its full affinity. The humanized1-17-2Ab could be used as vector for therapeutic vaccine. Also, through the example of1-17-2Ab humanization, we proposed a novel method based on epitopes scanning and MD simulation for antibody humanization.