Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice

Abstract

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

Keywords: antibody generation; immunoglobulin loci.

Fig. 1.

Sequential modifications leading to three versions of VelocImmune mice. Schematic representation (not to scale) of sequential modifications of the mouse IgH (A) and IgK (B) chain loci with increasing amounts human variable sequences. Double homozygous mice were made from three different stages of heavy and light chain humanization (C).

Fig. 2.

B-cell populations in WT and VelocImmune mice. Cells from spleen (A, C, and D) or inguinal lymph node (B) of WT or VelocImmune 1 (V1), 2 (V2), or 3 (V3) mice (column headings) were stained for surface IgM expressing B cells (A and B), surface Ig containing either kappa or lambda light chains (C), or surface IgM of specific allotypes (D); populations are separated by FACS.

Fig. 3.

Serum Ig levels. Levels of serum Ig in WT (white bars) or VelocImmune mice (gray bars) for IgM and IgG isotypes (A), as well as IgA (B) and IgE (C).