A tool kit for rapid cloning and expression of recombinant antibodies.

Tihomir S Dodev,Tihomir S Dodev,Sophia N Karagiannis,Sophia N Karagiannis,Holly Bowen,Andrew J Beavil,Marie O Pang,Debra H Josephs,Debra H Josephs,Debra H Josephs,Rebecca Beavil,Louisa K James,Amy E Gilbert,Amy E Gilbert,Panagiotis Karagiannis,Panagiotis Karagiannis,Hannah J Gould,Heather J Bax

doi:10.1038/srep05885

Abstract

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.

Highlights

Over the last four decades, molecular cloning has evolved tremendously
Over the last three decades, recombinant monoclonal antibodies (mAbs) have become a key tool for basic research, diagnosis and treatment of human diseases
An ideal antibody cloning method would require the minimum costs of labor and reagents and deliver high success rate of correctly assembled expression vectors

Summary

Introduction

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. Conventional methods for recombinant expression of whole antibodies of interest typically require the establishment of cell lines derived from CHO, mouse myeloma, or PER.C6 cells[19,20,21,22,23,24] This tends to be a lengthy, low efficiency process involving extensive selection and screening and is unfavourable for expressing large numbers of antibodies for functional studies, as may be required after generating a variable gene library. Matched Ig heavy- and light-chain V-regions of desired specificity have been amplified from single B cellderived cDNA and linked to the desired Ig constant (C) region in separate vectors encoding either the heavy- or light-chain DNA Transient transfection of these vectors in mammalian HEK293 cells has enabled the rapid production of recombinant mAbs[12,25,26,27,28]. We aimed to minimize the cost and time required to clone antibody heavy and light chains into a single mammalian expression vector for stable transfection of a suitable cell line, allowing an scalable production process and delivering sufficient material for pre-clinical studies

Objectives

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Conclusion