Rapid generation of specific antibodies by enhanced homologous recombination

Abstract

In the chicken immune system, gene conversion, a type of homologous recombination, primarily contributes to diversification of the immunoglobulin gene. Here, we report on the rapid generation of specific monoclonal antibodies using the chicken DT40 B-cell line undergoing gene conversion. We discovered that the gene conversion frequency at the immunoglobulin locus is increased by treating DT40 cells with a histone deacetylase inhibitor, trichostatin A (TSA), thereby generating diversity at the immunoglobulin locus in the majority of treated cells. This indicates that TSA treatment accelerates the autonomous diversification of surface IgMs on DT40 cells. We took advantage of this effect to select DT40 cells producing specific antibodies with antigen-conjugated magnetic beads. This autonomously diversifying library (ADLib) selection system enables the quick establishment (approximately 1 week from a diversifying library) of various clones producing monoclonal IgMs with enough specificity and affinity for immunological assays, and is applicable to various biotechnologies including rational protein design.

Noncompetitive Immunoassay Detection System for Haptens on the Basis of Antimetatype Antibodies

“…To overcome such limitations, we used the ADLib ex vivo antibody development system to establish antimetatype antibodies. Antibody diversification in this system is automatically promoted by DNA recombination in the antibody locus, which provides this system with the potential to generate antibodies with various specificities independently of antigen stimulation (22,23 ). This system also enables rapid antibody selection by affinity isolation like the magnetic beads-based immunoprecipitation method, which is supposed to avoid undesirable dissociation of the hapten-antibody immunocomplexes when used as bait.…”

“…We established antimetatype chicken mAbs that reacted with hapten-mAb complexes using the Autonomously Diversifying Library (ADLib) system (22,23 ). With the ADLib system, in general, the antibodies were isolated ex vivo from antibody libraries established by activating immunoglobulin gene diversification of chicken-derived DT40 cells, and the reactivity of candidate antibodies was screened by ELISA with antigen and horseradish peroxidase-conjugated antichicken antibodies.…”

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies

“…This approach makes possible both de novo discovery and maturation from a naïve antibody library and may also be used for maturation of preexisting antibodies. In vitro affinity maturation by SHM has been described in human B cell lymphoma lines (32), chicken DT40 cells (33,34), CHO cells (4), and 18-81 cells (35). However, these cell lines are difficult to transfect at efficiencies suitable for use with diverse libraries.…”

The chicken Ig light chain 3′‐enhancer is essential for gene expression and regulates gene conversion via the transcription factor E2A

“…The steady state level of the Ig RNA would therefore not change. However, this is unlikely since treatment of clone 18 cells with trichostatin A increases GC with a concomitant increase in transcription [35], suggesting that increased rates of transcription would be detectable in DT40 clone 18 cells. We therefore favour a model whereby the recombination in GC can be activated independently of transcription.…”