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

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.

Monoclonal antibody classification based on epitope-binding using differential antigen disruption.

Currently, classifying a population of specific antigen-reactive monoclonal antibodies (mAbs) according to their epitope-binding properties has been limited to competition assays. Such assays are time consuming, labor intensive and restricted to the number of mAbs in the experiment. To overcome this problem, a differential antigen disruption-based antibody profiling procedure was developed. This procedure rapidly classifies specific antigen-reactive mAbs into epitope-related groups by measuring the binding signal of the antibodies to a set of structurally disrupted antigens and then clustering the antibodies according to the similarity of their binding profiles. The clustering results generated by differential antigen disruption showed a significant concordance with those generated by competition experiments. Therefore, differential antigen disruption method opens an opportunity to assess the entire population of antigen-reactive mAbs according to their epitope-binding properties. In doing so, a set of representative antibodies can be drawn to describe the epitope complexity for systematically exploring their functions.