Human Antibodies by Guided Selection

Guided selection is a method for humanization of pre-existing non-human (for example, mouse) antibodies, based on chain shuffling of V-genes by using phage display technology. Mouse VH and VL domains are used to guide the selection of a human antibody partner and are replaced sequentially or in parallel with human VH and VL domains, respectively to derive human antibody. The resulting human antibodies therefore bind to epitopes that are at least overlapping with that of original mouse antibody. This chapter will describe the sequential guided selection procedure using a Fab format as an example. The first step is to clone the variable regions of a mouse antibody to a phagemid vector containing human constant domains, C? and CH1, which results in a chimeric Fab (see Subheading 3.1). The next step is to construct a human VL shuffled library by replacing the mouse VL with human VL repertoires (see Subheading 3.2). The third step is to select binders after panning against a target antigen (see Subheading 3.3), which results in selection of human VL paired with the mouse VH. The fourth step is to construct a human VH shuffled library by replacing the mouse VH in the semi-human Fab with human VH repertoires, similar to Protocol for VL shuffling. The fifth step is to select complete-human Fab clones after panning, similar to Protocol for the selection of human VL. The final step is to confirm the epitope specificity and affinities of selected human Fabs using proper assays. The final product after guided selection can be completely of human origin unlike other humanization methods and hence better therapeutic efficacy can be expected by removing problems related to the immunogenicity of mouse residues. In addition, the guided selection method can generate diverse human antibodies with better properties compared with original mouse antibody.