Rabbit Immune Repertoires for Generation and Humanization of Therapeutic Monoclonal Antibodies? 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. Rabbits with the rare b9 allotype are excellent sources for therapeutic monoclonal antibodies. We generated rabbit polyclonal antibodies from these rabbits. Broadly neutralizing antibodies against receptor-activated epitopes on the N-heptad repeat region (NHR) of HIV-1 gp41 are elicited during natural infection and by immunization. Our collaborator Dr. Michael Zwick rescued HIV-1-neutralizing anti-NHR antibodies from immune phage display libraries that were prepared from an HIV-1 infected individual, and from b9 rabbits that we immunized with a mimetic of the NHR inner trimeric coiled-coil previously described by Dr. C. Bewley, NIDDK. The human and rabbit monoclonal antibodies bind to overlapping epitopes on the NHR trimer, generate distinct binding profiles against a panel of gp41 mimetic proteins and neutralize primary HIV-1 from various clades with modest potency using a pseudotyped virus assay. An examination of the differing abilities of the antibodies to neutralize different viruses bearing identical NHR sequences has revealed a particular restriction to neutralization that appears to be steric in nature, rather than dependent on viral entry kinetics. Taken together, the results indicate that broadly neutralizing antibodies, albeit currently of limited potency, are elicited against the NHR trimer of HIV-1 gp41 during natural infection and by immunization. For vaccine design, the newly selected antibody panel can be used in optimizing the NHR trimeric coiled-coil to favorably display neutralizing epitopes (Nelson, J. D., Jensen, R., Bewley, C. A., Brunel, F. M., Louis, J. M., Clore, G. M., Mage, R. G., Dawson, P. E., Burton, D. R., and Zwick, M. B. 2008 (1)? The laboratory of Dr. C. Rader (NCI) isolated monoclonal antibodies selective for all three members of the Nogo-66 receptor (NgR) family. NgR family members are cell surface proteins involved in the development, plasticity, and regeneration of the central nervous system. NgR1, NgR2, and NgR3 are primarily expressed by neurons in the central nervous system (CNS) and believed to limit axonal growth and sprouting following CNS injury. In an attempt to define the expression and decipher the function of individual members of the Nogo-66 receptor family, we generated selective rabbit polyclonal antibodies (Venkatesh K, Chivatakarn O, Lee H, Joshi PS, Kantor DB, Newman BA, Mage R, Rader C, Giger RJ, The Nogo-66 receptor homolog NgR2 is a sialic acid-dependent receptor selective for myelin-associated glycoprotein. J Neurosci. 25:808-22, 2005). We then used phage display technology to generate rabbit monoclonal antibodies (mAbs) from the same immune repertoires. We obtained mAbs with nanomolar affinity to epitopes that are specific for NgR1 and NgR2, respectively, but at the same time conserved between mouse, rat, and human orthologs. Employing phage display vector pC3C, a newly designed phagemid optimized for the generation and selection of Fab libraries with human constant domains, rabbit mAbs were selected from chimeric rabbit/human Fab libraries, characterized in terms of specificity, affinity, and amino acid sequence, and finally converted to chimeric rabbit/human IgG. Strong and specific recognition of cell surface bound Nogo-66 receptor family members by chimeric rabbit/human IgG was demonstrated using immunofluorescence microscopy and immunoprecipitation. The rabbit mAbs together with their amino acid sequences constitute a defined panel of species cross-reactive reagents in infinite supply for investigations toward a functional role of the Nogo-66 receptor family in and beyond the CNS (1) and Venkatesh K, Raiker S, Lee A, Lee-Osbourne J, Wychowski T, Hofer T, Mage R, Rader C, and Giger RJ, Development of Nogo Receptor Variants with enhanced Ligand Binding Properties: Insights into the structural basis of the Myelin Associated Glycoprotein Nogo Receptor 2 association-- manuscript in preparation. In a collaboration with Drs. Wingfield, Stahl and Watts, we immunized b9 rabbits with recombinant HIV rev and HBeAg (Core e-antigen of Hepatitis B) for phage display and selection of Fab against conformational epitopes to be used for cocrystallization. Libraries generated from these immunized rabbits have been screened and Fab of interest recovered and sequenced.? The above studies derive from work that characterized rabbit pre-immune and post-immune repertoire development.? We studied genes of the rabbit immune system using techniques of molecular biology and immunology. In species such as mouse and human, generation of combinatorial diversity through use of different VH and VL genes in immunoglobulin VHDJH and VLJL rearrangements can be a major contributor to the primary antibody repertoire. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal as only a few VH genes are rearranged and expressed. This resembles chicken antibody formation. Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. As in the chicken, the 3-prime most VH1 gene is rearranged in most rabbit B lymphocytes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and also to secondary diversification during immune responses in germinal centers (GCs)(Mage RG, Lanning D, Knight KL,B cell and antibody repertoire development in rabbits: The requirement of gut-associated lymphoid tissues. Dev Comp Immunol 30:137-53, 2006).

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Hofer, Thomas; Tangkeangsirisin, Wisit; Kennedy, Michael G et al. (2007) Chimeric rabbit/human Fab and IgG specific for members of the Nogo-66 receptor family selected for species cross-reactivity with an improved phage display vector. J Immunol Methods 318:75-87
Mage, Rose G; Lanning, Dennis; Knight, Katherine L (2006) B cell and antibody repertoire development in rabbits: the requirement of gut-associated lymphoid tissues. Dev Comp Immunol 30:137-53
Venkatesh, Karthik; Chivatakarn, Onanong; Lee, Hakjoo et al. (2005) The Nogo-66 receptor homolog NgR2 is a sialic acid-dependent receptor selective for myelin-associated glycoprotein. J Neurosci 25:808-22
Popkov, Mikhail; Jendreyko, Nina; Gonzalez-Sapienza, Gualberto et al. (2004) Human/mouse cross-reactive anti-VEGF receptor 2 recombinant antibodies selected from an immune b9 allotype rabbit antibody library. J Immunol Methods 288:149-64
Mehr, Ramit; Edelman, Hanna; Sehgal, Devinder et al. (2004) Analysis of mutational lineage trees from sites of primary and secondary Ig gene diversification in rabbits and chickens. J Immunol 172:4790-6
Popkov, Mikhail; Mage, Rose G; Alexander, Cornelius B et al. (2003) 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. J Mol Biol 325:325-35
Sehgal, Devinder; Obiakor, Harold; Mage, Rose G (2002) Distinct clonal Ig diversification patterns in young appendix compared to antigen-specific splenic clones. J Immunol 168:5424-33
Obiakor, Harold; Sehgal, Devinder; Dasso, Joseph F et al. (2002) A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ. Anal Biochem 306:55-62
Sehgal, D; Schiaffella, E; Anderson, A O et al. (2000) Generation of heterogeneous rabbit anti-DNP antibodies by gene conversion and hypermutation of rearranged VL and VH genes during clonal expansion of B cells in splenic germinal centers. Eur J Immunol 30:3634-44
Dasso, J F; Obiakor, H; Bach, H et al. (2000) A morphological and immunohistological study of the human and rabbit appendix for comparison with the avian bursa. Dev Comp Immunol 24:797-814

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