From rabbit antibody repertoires to rabbit monoclonal antibodies

Abstract

In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies.

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility

“…43 In addition to V H -D-J H and V L -J L recombination and somatic hypermutation, rabbits extensively use somatic gene conversion for the diversification of antibody sequences 2 . Importantly, both somatic gene conversion and somatic hypermutation evolve the neonatal B-cell repertoire to the primary B-cell repertoire during the first two months after birth in gut associated lymphoid tissue (GALT).…”

“…Considering the extensive antibody sequence diversification in the primary B-cell repertoire, we argued that naïve rabbits would provide an outstanding source for mAbs that bind human antigens with high affinity and specificity. We generated a naïve rabbit antibody library that was designed to further increase the complexity of antibody sequences by (i) harvesting spleen and bone marrow from nine rabbits belonging to two different strains and housed at two different facilities, (ii) RT-PCR amplifying V H , V κ , and V λ sequences with an expanded set of primers in approximately 2,000 individual reactions to overcome natural biases due to, e.g., dominant usage of the V H 1 gene in V H -D-J H recombination, 43 (iii) randomly combining V H /V κ and V H /V λ sequences to generate a total of >10 10 independent antibodies. NGS analysis of the V H component of the naïve rabbit antibody library revealed a highly diverse antibody collection that retained key features of natural rabbit antibodies with respect to HCDR3 length distribution and amino acid sequence composition.…”

“…This property makes these mAbs particularly attractive for preclinical investigations in mouse models of human cancer, where the presence or absence of on-target-off-tumor toxicities of candidate mAbs can provide important information prior to clinical translation. 43 Collectively, our new panel of fully sequenced and extensively characterized 13 rabbit anti-human ROR1 mAbs and 12 rabbit anti-human ROR2 mAbs substantially expands the collection of antibody reagents available for studying and interrogating this family of receptor tyrosine kinases.…”

“…In breast cancer, they could provide treatment options for patients who do not benefit from HER2-targeting antibody therapeutics. Using established methods, 43 we humanized both XBR1-402 and XBR2-401 to further facilitate clinical translation (data not shown).…”

Rapid detection of three rabbit pathogens by use of the Luminex x-TAG assay

“…Domestic rabbit (Oryctolagus cuniculus), especially New Zealand white rabbit, has attracted more and more attention in biomedical, immunological and pharmaceutical research, because of its intermediate size and phylogenetic proximity to primates [1]. It played an important role in production of antibodies, eye research as well as cardiovascular disease [2,3]. Rabbit is one of the most commonly used experimental animals and must be free of some important pathogens.…”