Elsevier

Journal of Biotechnology

Volume 152, Issue 4, 10 April 2011, Pages 159-170
Journal of Biotechnology

A human scFv antibody generation pipeline for proteome research

https://doi.org/10.1016/j.jbiotec.2010.09.945Get rights and content

Abstract

The functional decryption of the human proteome is the challenge which follows the sequencing of the human genome. Specific binders to every human protein are key reagents for this purpose. In vitro antibody selection using phage display offers one possible solution that can meet the demand for 25,000 or more antibodies, but needs substantial standardisation and minimalisation. To evaluate this potential, three human, naive antibody gene libraries (HAL4/7/8) were constructed and a standardised antibody selection pipeline was set up. The quality of the libraries and the selection pipeline was validated with 110 antigens, including human, other mammalian, fungal or bacterial proteins, viruses or haptens. Furthermore, the abundance of VH, kappa and lambda subfamilies during library cloning and the E. coli based phage display system on library packaging and the selection of scFvs was evaluated from the analysis of 435 individual antibodies, resulting in the first comprehensive comparison of V gene subfamily use for all steps of an antibody phage display pipeline. Further, a compatible cassette vector set for E. coli and mammalian expression of antibody fragments is described, allowing in vivo biotinylation, enzyme fusion and Fc fusion.

Introduction

To date, antibodies represent the fastest growing class of biological therapeutics on the market and are indispensable as detection reagents in research and diagnostics (Dübel, 2007, Hagemeyer et al., 2009, Nieri et al., 2009). For proteome research, antibodies or alternative scaffolds are a key tool for the decryption of the human proteome (Aebersold and Mann, 2003, Berglund et al., 2008, Dübel et al., 2010, Hust and Dübel, 2004, Ohara et al., 2006, Skerra, 2007, Taussig et al., 2007, Wingren et al., 2009). After sequencing of human genomes, including the genome of individual persons (International Human Genome Sequencing Consortium, 2004, Levy et al., 2007, Wheeler et al., 2008) is well established, the research focus shifted to the analysis of gene products. The estimated number of protein encoding human genes is about 20,000–25,000 (International Human Genome Sequencing Consortium, 2004, Levy et al., 2007), due to alternative mRNA splicing variants and post-translational modifications including glycosylations, phosphorylation, sulfation etc. the number of different human proteins and protein variants is supposed to exceed the size of the genome severalfold (Harrison et al., 2002). The development of highly parallelized protein and tissue microarrays may revolutionise the proteome analysis in the same way nucleic acid microarrays accelerated the genome and transcriptome analysis (Stoevesandt et al., 2009).

For the human proteome atlas (www.proteinatlas.org), affinity purified polyclonal antibodies directed against protein expression signature tags (PRESTs) were generated in substantial number (Berglund et al., 2008). However, polyclonal antibodies are only a limited resource and without guarantee of constant quality. This can be overcome by the use of monoclonal antibodies. However, in respect of throughput, and despite becoming streamlined and automated to a considerable extent (De Masi et al., 2005), hybridoma technology still requires immunising of individual animals. Further, the immune system still defines the minimal time to obtain an immune response, whereas in vitro methods allow selection within a few days. Both, polyclonal and hybridoma technologies, do not provide direct access to the antibody gene sequence. Hence, subsequent genetic modifications to improve properties (e.g. affinity, stability etc.) or assay compatibility (e.g. fusion to alternative tags, switch between mono-, bi- or multivalency) are laborious. An added value is provided by the in vitro antibody selection methods which supply the antibody gene right from the start, for example to achieve functional knock down phenotypes of the protein bound by the antibody (Böldicke, 2007, Strebe et al., 2009).

In vitro technologies have demonstrated their potential to meet the demands of larger scale antibody generation projects (Deflorian et al., 2009, Dübel et al., 2010, Hallborn and Carlsson, 2002, Hust and Dübel, 2004, Konthur et al., 2005, Krebs et al., 2001, Mersmann et al., 2010, Ohara et al., 2006, Taussig et al., 2007), and still offer much potential to be further shortened and miniaturised, which mice and rabbits cannot. Here, the full potential of other in vitro selection display technologies, e.g. ribosome display (Hanes and Plückthun, 1997, He and Taussig, 2008), remains to be explored as well.

In the last two decades the development of phage display was driven mainly by the opportunity to make human antibodies for therapeutic applications. However, the technology is proven to yield valuable research tools, which start to appear on the market (Zitat: http://www.axxora.com/scripts/pdf/pdf_megadetail.php3?PID=AG-27B-0001). To achieve that, high efforts were invested in the generation of a large number (i.e. “as many as possible”) of antibody candidates against few well-characterised antigens (Dübel, 2007, Hoogenboom, 2005, Taussig et al., 2007, Thie et al., 2008). In contrast, for research antibodies or alternative scaffolds have to be generated against thousands of poorly characterised proteins, many of them not yet known at all. Consequently, an integrated process combining antibody selection, screening and production which is amenable to parallelisation and miniaturisation as well as to the integration of different antigen sources, like peptides, proteins, fusion proteins, protein, protein complexes, PRESTs, is required (Dübel et al., 2010, Hust and Dübel, 2004, Konthur et al., 2005, Taussig et al., 2007).

The generation and application of very large standardised universal ‘single pot’ antibody gene libraries, which in principle contain binders against every possible antigen, is a key to the success of an universal antibody generation pipeline. Therefore, a naive antibody gene library was constructed for a simplified antibody selection pipeline designed to be able to generate antibodies for proteome research for reasonable cost. Beyond the use in research, since it employs a human antibody repertoire, antibodies selected from this library may further be developed for use in diagnostics and therapy with little effort. The quality of the library and process was validated by the selection of binders to 110 antigens.

Section snippets

Cell lines and bacterial strains

E. coli XL1-Blue MRF′ (Stratagene, Amsterdam, NL) (F′::Tn10(Tetr) proAB+ lacIq Δ(lacZ)M15/recA1 endA1 gyrA96 (Nalr) thi hsdR17 (rK–mK+) glnV44 relA1 lac) was used for antibody gene library construction, phage display and screening.

The transformed human embryonic kidney (HEK) cell line 293T (American Type Culture Collection, ATCC, Rockwell, MD, No. CRL-11268) was cultured in DMEM (4.5 g/L glucose) supplemented with 2 mM l-glutamine, 10% (v/v) fetal calf serum (FCS) and 1% (v/v)

Construction of the antibody gene libraries HAL 4/7/8

The first step in the generation of an universal antibody selection pipeline (Fig. 1) is the generation of a large antibody gene library using a phagemid which allows phage display, selection and screening without any subcloning steps. For the construction of the human naive antibody gene libraries HAL4/7/8, lymphocytes were isolated from the blood of 44 donors of Caucasian, African, Indian and Chinese origin. Total RNA and mRNA were isolated from the isolated lymphocytes and reverse

Discussion

Various methods have been applied to clone the genetic diversity of antibody repertoires. Naive or immune antibody gene libraries are mostly constructed by one, two or three separate cloning steps. In the “one cloning step” method VH and VL are combined by assembly PCR and the PCR product is cloned into the vector (Clackson et al., 1991, McCafferty et al., 1994, Vaughan et al., 1996). In the “two step cloning” strategy, the amplified repertoire of light chain genes is cloned into the phage

Acknowledgments

We would like to thank Dr. Henk S.P. Garritsen (Inst. for Clinical Transfusion Medicine, Städtisches Klinikum Braunschweig) for kindly providing blood samples and support. We gratefully acknowledge the financial support by the German ministry of education and research (BMBF, SMP “Antibody Factory” in the NGFN2 program), the German Research Foundation (DFG) Grant DU 337/3-1, the EU FP6 supported coordination action funded activities “Proteome Binders” (contract 026008) and the EU FP7

References (66)

  • M. Little et al.

    Generation of a large complex antibody library from multiple donors

    J. Immunol. Methods

    (1999)
  • G.A. Løset et al.

    Construction, evaluation and refinement of a large human antibody phage library based on the IgD and IgM variable gene repertoire

    J. Immunol. Methods

    (2005)
  • C. Menzel et al.

    Human antibody RNase fusion protein targeting CD30+ lymphomas

    Blood

    (2008)
  • M. Mersmann et al.

    Towards proteome scale antibody selections using phage display

    New Biotechnol.

    (2010)
  • B.H. Nilson et al.

    Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain

    J. Biol. Chem.

    (1992)
  • A. Schmiedl et al.

    Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli

    J. Immunol. Methods

    (2000)
  • J. Shukla et al.

    Development and evaluation of antigen capture ELISA for early clinical diagnosis of chikungunya

    Diagn. Microbiol. Infect. Dis.

    (2009)
  • A. Skerra

    Alternative non-antibody scaffolds for molecular recognition

    Curr. Opin. Biotechnol.

    (2007)
  • G. Soltes et al.

    On the influence of vector design on antibody phage display

    J. Biotechnol.

    (2007)
  • N. Strebe et al.

    Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies

    J. Immunol. Methods

    (2009)
  • H. Thie et al.

    Multimerization domains for antibody phage display and antibody production

    New Biotechnol.

    (2009)
  • R. Aebersold et al.

    Mass spectrometry-based proteomics

    Nature

    (2003)
  • T. Böldicke

    Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER

    J. Cell. Mol. Med.

    (2007)
  • M. Cao et al.

    Construction, purification, and characterization of anti-BAFF scFv–Fc fusion antibody expressed in CHO/dhfr-cells

    Appl. Biochem. Biotechnol.

    (2009)
  • T. Clackson et al.

    Making antibody fragments using phage display libraries

    Nature

    (1991)
  • F. De Masi et al.

    High throughput production of mouse monoclonal antibodies using antigen microarrays

    Proteomics

    (2005)
  • S. Dübel

    Recombinant therapeutic antibodies

    Appl. Microbiol. Biotechnol.

    (2007)
  • J. Glanville et al.

    Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire

    Proc. Natl. Acad. Sci. U.S.A.

    (2009)
  • M.E. Goldsmith et al.

    Adsorption protein of the bacteriophage fd: isolation, molecular properties, and location in the virus

    Biochemistry

    (1977)
  • C.E. Hagemeyer et al.

    Single-chain antibodies as diagnostic tools and therapeutic agents

    Thromb. Haemost.

    (2009)
  • J. Hallborn et al.

    Automated screening procedure for high-throughput generation of antibody fragments

    Biotechniques Suppl.

    (2002)
  • J. Hanes et al.

    In vitro selection and evolution of functional proteins by using ribosome display

    Proc. Natl. Acad. Sci. U.S.A.

    (1997)
  • P.M. Harrison et al.

    A question of size: the eukaryotic proteome and the problems in defining it

    Nucleic Acids Res.

    (2002)
  • Cited by (0)

    1

    Present address: Novartis Institutes for BioMedical Research, Basel, Switzerland.

    2

    Present address: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.

    3

    Present address: Chemgineering Technology GmbH, Wiesbaden, Germany.

    4

    Present address: Sanofi-Aventis, Frankfurt, Germany.

    5

    Both senior authors contributed equally for this work.

    View full text