Humanization of the antibodies by germline-humanization

Humanization of the macaque antibodies by germline-humanization
The sequence of each antibody was compared with the human germline genes using the
IMGT/V-QUEST online tool from the International ImmunoGeneTics information system1
(IMGT) (http://www.imgt.org). This tool allows identification of the human germline genes
most similar to any given variable region and calculates the Germinality Index (GI), defined as
the percentage identity between a given framework region (FR) and the most similar human
germline sequence. Based on the physiochemical classes of the amino acids (AA), differences
in the FRs were classified as very similar, similar, dissimilar and very dissimilar AA. Firstly,
humanized variants of VH and VL were designed by exchanging the very similar AA and similar
AA followed by dissimilar AA and very dissimilar AA. The structure of each domain was
modeled using WAM antibody modeling [33] and visualized using UCSF Chimera software
[45]. The humanized VH and VL genes were synthesized by GeneArt1 Gene Synthesis
(Regensburg, Germany) and were used for expression of scFv-Fc and IgG.