Rabbit Immune Repertoires as Sources for Therapeutic Monoclonal Antibodies: The Impact of Kappa Allotype-correlated Variation in Cysteine Content on Antibody Libraries Selected by Phage Display Mikhail Popkov 1 , Rose G. Mage 2 * , Cornelius B. Alexander 2 Sujatha Thundivalappil 1 , Carlos F. Barbas III 1 * and Christoph Rader 1 * 1 Department of Molecular Biology and The Skaggs Institute for Chemical Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037, USA 2 Laboratory of Immunology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD 20892, USA The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. On the basis of phage display technol- ogy, we recently reported the first humanization of a rabbit monoclonal antibody. The allotypic diversity of rabbit immunoglobulins prompted us to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. In particular, we evaluated the diversity of unselected and selected chimeric rabbit/human Fab libraries that were derived from different kappa light chain allotypes. Most rabbit light chains have an extra disulfide bridge that links the variable and con- stant domains in addition to the two intrachain disulfide bridges shared with mouse and human kappa light chains. Here we evaluate the impact of this increased disulfide bridge complexity on the generation and selec- tion of chimeric rabbit/human Fab libraries. We demonstrate that rabbits with mutant bas and wild-type parental b9 allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones with b9 allotype is a rabbit/human Fab that binds with a dis- sociation constant of 1 nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer. Examination of 228 new rabbit antibody sequences allowed for a comprehensive com- parison of the LCDR3 and HCDR3 length diversity in rabbits. This study revealed that rabbits exhibit an HCDR3 length distribution more closely related to human antibodies than mouse antibodies. q 2003 Elsevier Science Ltd. All rights reserved Keywords: rabbit monoclonal antibodies; antibody engineering; antibody library; phage display; immunoglobulin kappa light chain *Corresponding authors Introduction Monoclonal antibodies (mAbs) are gaining importance in the treatment of various conditions, including cancer, cardiovascular diseases, and viral infections. In concert with their clinical accep- tance, mAbs have become commercially viable drugs. 1,2 Today, mAbs are generated by either hybridoma technology or from antibody libraries. 3 The development of antibody libraries has been greatly influenced by the development of display technologies and vice versa. The physical connec- tion of antibody phenotype (protein) and genotype (gene) effectively allows selection rather than screening of antibody libraries. The integration of antibody libraries and phage display technology 4 about a decade ago 5,6 was a key event in this 0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved E-mail addresses of the corresponding authors: rmage@niaid.nih.gov; carlos@scripps.edu; crader@scripps.edu Abbreviations used: mAb, monoclonal antibody; CDR, complementarity determining region; HCDR3, heavy chain complementarity determining region 3; LCDR3, light chain complementarity determining region 3; V, variable domain; C, constant domain; NZW, New Zealand White; ang-2, angiopoietin-2. doi:10.1016/S0022-2836(02)01232-9 J. Mol. Biol. (2003) 325, 325–335