Human antibody production in transgenic animals

Abstract

Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.

Fig. 1

Human IgH loci. a The complete V_H, D and J_H region from the most 5′ V_H, IgHV3–74, to the most 3′ J_H segment, J_H6, is accommodated on ~950 kb (Lefranc and Lefranc 2001). b Transgenic constructs and features. Animals were derived from manipulated fibroblasts (cattle) or ES cells (mouse) and by DNA microinjection (mouse and rat). In several lines, human Ig expression appeared to be reduced. Endogenous VDJ use or H chain products with non-human segments, such as mouse or cattle Vs, were obtained in strains with leaky or incomplete endogenous KO