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

Laure-Hélène Ouisse,Ignacio Anegon,Jonathan Visentin,Frank Halary,Michael Osborn,Marianne Bruggemann,Laetitia Gautreau-Rolland,Xavier Saulquin,Roland Buelow,Melinda Moyon,Marie-Claire Devilder

doi:10.1186/s12896-016-0322-5

Abstract

BackgroundThere 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.ResultsThis 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 tetramer-based 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).ConclusionsTherefore, 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.

Highlights

There is an ever-increasing need of monoclonal antibodies for biomedical applications and fully human binders are desirable due to their reduced immunogenicity in patients
We demonstrate the presence of naturally circulating peptide-major histocompatibility (pMHC)-specific B lymphocytes in all human peripheral blood samples tested and generated a human monoclonal antibodies (mAbs) against the HLA-A2/Pp65495 peptide complex derived from human CMV but that displayed of low affinity
Following a method previously described that yielded high affinity antibodies [18], human immunoglobulin transgenic rats were immunized at day 0 at each side of the tail base with 100 μg of protein in Complete Freund Adjuvant (CFA) and at day 16 with 100 μg of protein in PBS, lymph nodes and spleens were harvested at day 21

Summary

Introduction

There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are desirable due to their reduced immunogenicity in patients. Human recipient immune response against murine mAbs is an important obstacle to their use due to their rapid clearance [4, 5] To solve this problem, several strategies have been developed including the modification of antibody protein sequences to decrease immunogenicity, such as generation of chimeric mousehuman or humanized antibodies, these strategies increase the cost of production and often decrease their affinity [6]. One of them is to use human B or plasma cells [7, 8], this technique is restricted to antigens, such as infectious agents following natural infection, and excludes many important targets that are either normal constituents of the organisms and for which there is immune tolerance or antigens that are harmful if administered, such as toxins Another technique is the use of phage or yeast display but this generates antibodies with weak affinities, and strategies to increase affinity are costly, time consuming and not always successful. Current efforts have focused on the use of human mAbs that have reduced immunogenicity after injection in humans compared to chimeric or murine antibodies

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Conclusion