A human scFv antibody generation pipeline for proteome research
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
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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.