A highly functional synthetic phage display library containing over 40 billion human antibody clones.

Marcel Weber,Sarah Wulhfard,Mattia Matasci,Alessia Putelli,Alessandra Villa,Teresa Hemmerle,Laura Gualandi,Dario Neri,Emil Bujak,Mark Isalan

doi:10.1371/journal.pone.0100000

Abstract

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.

Highlights

Monoclonal antibodies represent an important class of pharmaceutical biotechnology products and important research tools in chemistry and in life sciences [1,2]
While libraries were initially created starting from antibody genes isolated from natural sources (e.g., B cells in peripheral blood, spleen and tonsils [6,7]), there has been a growing interest in the construction of rationally designed synthetic antibody libraries, in which individual library members incorporate structural features which are beneficial for practical applications [8]
Design and Cloning of the Antibody Library To generate a large, stable and highly diverse library of functional antibody in the single chain variable fragment format, we cloned single-chain fragment variable (scFv) fragments with sequence diversity restricted to the CDR3 loops of both heavy and light chain into a phagemid vector

Summary

Introduction

Monoclonal antibodies represent an important class of pharmaceutical biotechnology products and important research tools in chemistry and in life sciences [1,2]. While libraries were initially created starting from antibody genes isolated from natural sources (e.g., B cells in peripheral blood, spleen and tonsils [6,7]), there has been a growing interest in the construction of rationally designed synthetic antibody libraries, in which individual library members incorporate structural features which are beneficial for practical applications [8] Such libraries may yield clones which are homogenous in terms of their biophysical properties and amino acid sequence ( facilitating affinity maturation procedures [9]), with some additional desirable properties, such as protein A binding for affinity capture applications [10]. The three antibodies, which have been shown to selectively recognize stromal and neovascular structures in cancer [14,15] and inflammation [16], are able to preferentially localize at sites of pathological angiogenesis in vivo and are currently being investigated in Phase I and Phase II clinical trials [17,18,19]

Methods

Results

Conclusion