Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies
Abstract
Background: There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and
fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a
strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by
reconstruction of human mAbs by RT-PCR and expression cloning.
Results: This strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbs
against major histocompatibility complex molecules loaded with peptides (pMHC). We then implemented this
technology using human immunoglobulin transgenic rats, which after immunization with an antigen of interest
express high affinity-matured antibodies with human idiotypes. Using rapid immunization, followed by tetramerbased
B-cell sorting and expression cloning, we generated several fully humanized mAbs with strong affinities,
which could discriminate between highly homologous proteins (eg. different pMHC complexes).
Conclusions: Therefore, we describe a versatile and more effective approach as compared to hybridoma generation
or phage or yeast display technologies for the generation of highly specific and discriminative fully human mAbs that
could be useful both for basic research and immunotherapeutic purposes.
Domains
Cancer
Origin : Files produced by the author(s)