The Rabbit Antibody Repertoire as a Novel Source for the Generation of Therapeutic Human Antibodies

Abstract

The rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for the generation of therapeutic human antibodies. The humanization of rabbit antibodies, however, has not been reported. Here we use phage display technology to select and humanize antibodies from rabbits that were immunized with human A33 antigen which is a target antigen for the immunotherapy of colon cancer. We first selected rabbit antibodies that bind to a cell surface epitope of human A33 antigen with an affinity in the 1 nM range. For rabbit antibody humanization, we then used a selection strategy that combines grafting of the complementarity determining regions with framework fine tuning. The resulting humanized antibodies were found to retain both high specificity and affinity for human A33 antigen.The growing significance of antibody-based strategies for the treatment of a variety of diseases demands efficient and reliable routes to human or humanized antibodies with high specificity and affinity. Nonhuman antibodies are highly immunogenic in humans thereby limiting their potential use for therapeutic applications, especially when repeated administration is necessary. In order to reduce their immunogenicity, nonhuman antibodies have been humanized using strategies that are based on rational design, in vitro evolution, or a combination of both. An alternative route is the direct generation of human antibodies from transgenic mice containing human immunoglobulin (Ig) 1 loci or by selection from naive or synthetic human antibody libraries displayed on phage (1, 2).Here we exploit a new route to humanized antibodies. We use phage display technology to select and humanize antibodies from rabbits that were immunized with a human antigen. Monoclonal antibodies from multiple species are accessible by screening combinatorial antibody libraries displayed on phage (3). The accessibility of antibody repertoires of multiple species might become substantial in the search for new therapeutic antibodies. In particular the rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for therapeutic antibodies. The humanization of rabbit antibodies, however, has not been reported.To allow selective antibody targeting, both high specificity of the antibody and restrictive tissue expression of the antigen are required. For cancer therapy, only a few antibody-antigen systems that meet these requirements have been identified (4). The A33 system which has been established in clinical trials is one of them (5-7). Mouse monoclonal antibody A33 (5) detects an antigen that is expressed in normal human colon epithelial cells and by Ͼ95% of human colon cancers. Human A33 antigen (8, 9) is a transmembrane glycoprotein of the Ig superfamily. It consists of 2 extracellular Ig folds, a single transmembrane domain, and a highly polar intracellular tail containing a Spalmitoylation si...

Phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesisin vivo

“…Specific oligonucleotide primers (26,29,30) were used to amplify heavy chain variable domain and light chain variable domain gene segments from purified phagemid DNA of Fab VR05 and Fab 2S08b. Light chain variable domain segments of VR05 and 2S08b were amplified with ompseqgtg and RKB9Jo-BL.…”

Neutralization ofBurkholderia pseudomalleiProtease by Fabs Generated through Phage Display

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“…The antisense primer lead-B hybridizes to a sequence upstream of the Fd fragment coding sequence and is used to amplify the C coding sequence together with the sequence intervening light-and heavy-chain fragment coding sequences in phagemid vector pComb3H. 14) Based on this strategy, the chimeric mouse/human light-and heavy-chain fragment coding sequences were assembled and fused by two sequential overlap extension PCR steps. In the first step, mouse V L and human C were fused using the primer combination RSC-F (5 0 -gaggaggaggaggaggaggcggggcccaggcggccgagctc-3 0 )/ lead-B, and mouse V H and human C H 1 were fused using the primer combination lead-V H (5-gctgcccaaccagccatggcc-3 0 ) and dp-EX (5 0 -gaggaggaggaggaggagagaagcgtagtccggaacgtc-3 0 ).…”

A33scFv–cytosine deaminase: a recombinant protein construct for antibody-directed enzyme-prodrug therapy

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