Humanization is significant for reducing the immunogenicity of monoclonal antibodies derived from rodent sources and for improving their activation of the human immune system.
Rodent antibodies are highly immunogenic in humans, which limits their clinical applications, especially when repeated administration is required. Importantly, they are rapidly removed from circulation and can cause systemic inflammatory effects as well.
As a means of circumventing these problems, Creative Biolabs has developed three antibody humanization strategies that can preserve the specificity and affinity of the antibody toward the antigen whereas significantly or completely eliminate the immunogenicity of the antibody in humans. The first approach is CDR grafting and the second approach is chain shuffling. Both the two methods are based on phage display of humanized scFv variants and selection of high-affinity humanized binders through bio-panning. The third method, humanized IgG library screening, is somehow unique. Creative Biolabs’s scientists display humanized IgG library on the surface of mammalian cells and then high-affinity binders will be sorted by FACS. This article is mainly talking about the specialty of the third approach.
First, researchers select an acceptor human VH and a receptor VL from a subgroup of human antibodies based on consensus sequences. Next, CDR grafting is conducted. In order to increase the affinity of the humanized antibodies, a mammalian cell surface display IgG library is created to display all possible variants of the humanized IgG.
Since the size limit for the mammalian cell surface display IgG library, decisions must be made as to which amino acids to diversify and to what extent so that there are fewer nonsense antibody mutants that waste the capacity of the library. Back mutations in framework regions are designed based on computer modeling or antigen/antibody structure information. Also, scientists will distinguish between residues with solvent accessible side chains from those with buried side chains. Creative Biolabs has claimed that they confirmed that randomization of residues with buried side chains is a waste of library sequence space. Also, they do not mutate glycines and tryptophans since changes in these residues usually abolish binding. In order to create a largest possible library, trimer codon technology is employed to randomize those defined framework residues on both humanized heavy chain and humanized light chain.
This method allows selection of humanized antibodies in a full-size IgG format that retain the original affinity of the mouse antibody. In comparison with the commonly used bacterial phage display based methods, it allows selection of high affinity humanized antibodies in a dimeric IgG format; selected IgG are immediately good for further downstream applications—avoiding time consuming conversion of scFv to IgG and codon optimization of converted IgG, which sometimes deselect some humanized scFvs. In addition, this approach overcomes the problem that some antibodies have sequences that prevent protein synthesis and phage display in bacterial cells.
With the practical strategy, it turns out that antibody affinity maturation is an integrated step in Creative Biolabs’ humanization procedure, thus improving the affinity after humanization is not a necessity. Notably, Creative Biolabs also developed a proprietary in vivo approach to evaluate the immunogenicity of the humanized antibodies in primates for the purpose of mimicking the true immunogenicity of the humanized antibodies in humans.
Creative Biolabs, a leading life science organization that has very extensive experience in cell services and engineering fields, has always been highly recommended through the whole industry for its innovation. It is always willing to share and solve problems.
