Molecules 2011, 16, 412-426; doi:10.


ISSN 1420-3049 Review

Phage Display for the Generation of Antibodies for Proteome Research, Diagnostics and Therapy
Thomas Schirrmann, Torsten Meyer, Mark Schütte †, André Frenzel and Michael Hust * Technische Universität Braunschweig, Institute of Biochemistry and Biotechnology, Department of Biotechnology, Spielmannstr. 7, 38106 Braunschweig, Germany † Present address: Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany * Author to whom correspondence should be addressed; E-Mail: Received: 10 November 2010; in revised form: 4 January 2011 / Accepted: 7 January 2011 / Published: 10 January 2011

Abstract: Twenty years after its development, antibody phage display using filamentous bacteriophage represents the most successful in vitro antibody selection technology. Initially, its development was encouraged by the unique possibility of directly generating recombinant human antibodies for therapy. Today, antibody phage display has been developed as a robust technology offering great potential for automation. Generation of monospecific binders provides a valuable tool for proteome research, leading to highly enhanced throughput and reduced costs. This review presents the phage display technology, application areas of antibodies in research, diagnostics and therapy and the use of antibody phage display for these applications. Keywords: antibody; scFv; phage display; cancer; diagnostics; proteome research; panning

1. Polyclonal and Monoclonal Antibodies Production of polyclonal antibodies by immunisation of animals is a method established for more than a century. The first antibody serum was produced in horses and directed against diphtheria [1]. Hybridoma technology was the next milestone, allowing the production of monoclonal antibodies by fusion of an immortal myeloma cell with an antibody-producing spleen cell [2]. However, hybridoma technology has some limitations: possible instability of the aneuploid cell lines, most of all its limited

17]. First.5]. Production of immunoglobulin G (IgG) in E. most of the successful systems uncouple antibody expression from phage propagation by providing the genes encoding the antibody::pIII fusion proteins on a separate plasmid (called “phagemid”). The alternative is antibody generation using transgenic mice or cows which comprise human immunoglobulin loci instead of murine Ig loci. this technology is also restricted due to ethical reasons. This phagemid contains a phage morphogenetic signal for packaging the vector into the assembled phage particles. The first antibody gene repertoires in phage were generated and screened by using the lytic phage Lambda [16. these transgenic animals produce human antibodies [11-15]. The immunogenicity can be reduced by chimerisation or humanisation [6-10]. In our opinion.g. Antibody Phage Display An alternative for the generation of human antibodies is antibody phage display. coli is only possible in rare cases [27. two different genetic systems have been developed. but in our opinion the immunisation of animals does not allow generation of antibodies for proteome projects. 16 413 possible application to generate human antibodies and its inability to provide antibodies against toxic or highly conserved antigens [3]. the antibody gene replication and expression is uncoupled from the phage replication cycle. infected or immunised humans.. In the same way. Using the phagemid system. Therefore. For the expression of antibody::pIII fusion proteins for phage display. Due to limitations of the E. . antibody genes have been directly inserted into the phage genome fused to the wildtype M13 phage protein III (pIII) gene [20]. This technology can generate antibodies for a limited amount of therapeutic targets. There are two possibilities for the generation of human antibodies using hybridoma technology: the first is the generation of human hybridomas. Here. the IgG phage display cannot compete with Fab or scFv display and the additional Fc part is not a benefit for the display of binders. the variable heavy domain of camels (VHH) or humans (dAbs). only antibody fragments like single chain fragment variable (scFv). because of the effort required to immunise and handle thousands of animals. Smith [18] on filamentous phage display. Hence. however with limited success. However. The resulting peptide::pIII fusion protein is displayed on the surface of phage allowing affinity purification of the peptide and its corresponding gene. leading to a higher genetic stability and a simplification of the antibody gene library amplification. which bind specifically without a corresponding light chain variable domain. the genotype and phenotype of oligo-peptides were linked by fusing the corresponding gene fragments to the minor coat protein III gene of the filamentous bacteriophage M13. which is completely independent from any immune system and uses an in vitro selection process. This experimentally very difficult technology depends on the availability of B-lymphocytes from e. because repeated administration can cause human anti-mouse antibody reaction (HAMA) [4.28].21-23]. The display method most commonly used today is based on the ground breaking work of Georg P. antibody fragments fused to pIII can be presented on the surface of M13 phage [19-23]. fragment antigen binding (Fabs).Molecules 2011. After immunisation. are used routinely for antibody phage display [24-26]. 2. Murine antibodies are limited regarding a therapeutic application. coli folding machinery. a helper phage is needed for the production of antibody phage particles [19.

Selection of Antibodies 414 In vitro isolation of antibody fragments from antibody gene libraries by their binding activity is called “panning”.. Figure 1. competitor) parameters can be controlled to select antibodies able to bind the antigen under these defined conditions. 16 3. are necessary to select specifically binding antibody fragments. pH) and biological (e.g.g.. soluble monoclonal antibody fragments or antibody phage are produced and specific antigen binding is . Subsequently. Specifically binding antibody phage are eluted (e. coli. Usually two or three panning rounds. temperature). coli are infected with a helperphage to produce new antibody phage which can be used for further panning rounds until a significant enrichment of antigen specific phage is achieved. by trypsin or pH shift) and reamplified by infection of E. After panning. During this incubation step. The antigen is immobilised on a solid surface such as nitrocellulose [30].. chemical (e. the phagemid bearing E. physical (e. referring to the gold washer‘s tool [29] (Figure 1). magnetic beads [31].g. Antibody phage particles which bind weakly to the antigen and the vast excess of non-binding antibody phage are removed by stringent washing.Molecules 2011. column matrices [19]. Schematic overview of the selection of antibodies (“panning“) by phage display. and sometimes up to six.g.. plastic surfaces like polystyrene tubes [32] or 96 well microtitre plates [23] and incubated with antibody phage of the antibody gene library.

. [48] and the HAL scFv libraries [40]. the antibody – depending on the desired application – can be reconverted into different antibody formats (e. of the antibodies selected by phage display can be increased by additional in vitro affinity maturation steps [41-43]. at least in theory [47]. originated from autoimmune patients [25]. but always the CDR H3.40].33-36]. 16 415 analysed by ELISA to identify individual binders. In this library.. 5. The first antibodies derived from phage display for basic research have reached the market (e.g. Because the gene sequence of the binder is available. V-genes of these libraries contain hypermutations and are affinity matured. or more recent microarrays [60. purification of molecules or cells by affinity chromatography [59]. anti-CD256 from Axxora).000 protein encoding genes [62-64]. Semi-synthetic libraries are constructed from unrearranged V genes from pre B cells (germline cells) [49] or from one antibody framework [50] in which one or several complementary determining regions (CDR). To date. Due to alternative mRNA splicing and post-translational modifications.52]. Fully synthetic libraries are made of human frameworks with randomised CDR cassettes [51. flow cytometry [58]. are randomised. By different interpretation of the same genetic content.g.000-25. dramatic morphological . light chains from autoimmune patients were combined with a Fd fragment containing synthetic CDR1 and CDR2 in the human VH3-23 framework and naive CDR3 regions. Naive libraries are constructed from rearranged V genes from B cells (IgM) of non-immunized donors. e. these individual binders can be sequenced and further biochemically characterised [3. Antibodies for Basic Research Antibodies are essential tools for biological basic research. ELISA [56].61]..Molecules 2011. Affinity.g. Examples for this library type are the naive human Fab library constructed by de Haard et al. as they are designed to isolate antibody fragments binding to every possible antigen. Naive.. different types of antibody gene libraries can be constructed.g. After sequencing of the human genome. Afterwards. glycosylation. [55]. phosphorylation etc. 4. including the genome of individual persons. An important field for antibodies in basic research is proteome research. the number of different human proteins is supposed to exceed the amount of genes severalfold [65]. scFv-Fc fusion or IgG) and produced in different production hosts [39. This panning process can also be performed in a high-throughput manner [36-38]. Many methods require antibodies to specifically detect proteins or other molecules. the research focus shifted to the analysis of gene products. immunofluorescence microscopy [57]. Immune libraries are constructed from immunised donors and typically generated and used in medical research to get an antibody against one particular target antigen. of an infectious pathogen [21]. semisynthetic and synthetic libraries have been subsumed as “single-pot” libraries. Antibody Gene Libraries For scientific or medical applications. The human genome encodes about 20. but also the stability. A combination of naive and synthetic repertoire was used for the FAB310 antibody gene library. “single-pot” antibody libraries with a theoretical diversity of up to 1011 independent clones have been generated [53] to serve as a molecular repertoire for phage display selection procedures. e. Often used standard methods are immunoblot [54].. [44-46].

Other prominent technologies used for diagnostics are lateral flow strip assays [81. there are many examples for the application of antibodies derived by phage display for diagnostics [92-94].82].g. Here. localisation and function of its gene products. Therefore. and more recently.90]. Here. flow cytometry [58.. The advantage of an antibody competition assay is its species independence [80]. e. e. Antibody phage particles can also be used for immuno-PCR [91].84]. 28 therapeutic monoclonal antibodies were approved by the FDA and for further approvals are pending (US Food and Drug Administration) ( [73].. bead based assays such as Luminex [83] [72] while information about analysed human proteins are given in the “Human Proteome Atlas” (www. 16 416 variations are achieved. . e. between caterpillar and butterfly. The first way is the detection of antigens using antibodies [77] and the second. for each gene tools are necessary to investigate amount.g.g.70. but reversed way is the detection of serum antibodies using antigens [78]. In comparision to the immunisation of animals. the hemagglutination assay [85]. biotinylated antibody or scFv-phoA fusion [40] to the desired diagnostic assay. allowing a fast adaption of the antibody format e.. the storage and the provision of the binders can be achieved by storage of the corresponding DNA sequence which is directly available. To date.landesbioscience. Antibodies for Therapeutical Applications In 2010. as the genotype defines the species of an organism. only in vitro technologies like phage display or ribosome display [74] can fulfill the requirement to generate binders like antibody fragments or alternative binders e. pilot projects to generate antibodies to the complete human proteome were performed and complete antibody generation pipelines were developed [40.g. A big advantage of antibody phage display for diagnostics is the direct access to the genetic information of the binder. Sensitivity of the diagnostic assay can be enhanced by detection of the about 2700 copies of pVIII of an antibody phage by secondary antibodies [89. e. in vitro technologies are automatable [36-38]. 7. here. The information about individual binders against human proteins are stored and shared in the “Antibodypedia” project (www. Antibodies for Diagnostics There are three general ways to use antibodies for diagnostics.. the proximity ligation assay [86] or molecular imaging [87]. DARPins [75] or anticalins [76] to the complete human proteome.proteinatlas.71]. In our opinion. antibodies are a key tool for the decryption of the human proteome [47. An interesting diagnostic method is the direct use of phage particles fused to antibodies [88] for diagnostics.Molecules 2011. from pathogens [95-97]. but the proteome defines the phenotype. in vitro technologies can deliver an unlimited and defined resource of binders. 6. Mechanisms of therapeutic antibodies are manifold and include neutralisation of substances. For this purpose. scFvFc.66-69]. Most of the accepted therapeutic antibodies are for cancer and autoimmune diseases [98].g. IgG.g. The third way is the competition mabs/about). Furthermore. Combining antibody phage display with antigen phage display all steps from identification of antigens to the generation of antibodies against these antigens for diagnostics are available. Phage display allows also the identification of immunogenic proteins.antibodypedia. antigens are detected by serum antibodies which compete with a defined antibody preparation [79].

antibodies were humanised. Afterwards. The anticipated approval date for Benlysta by the US Food and Drug Administration is 2011 (www.fda. or combinations of these effects [102]. Later. This antibody was still of murine origin [101]. binding to cells and modulating the host immune system [101].99]. also the murine framework regions of the variable domains were replaced by their human counterpart. The next therapeutic antibodies were chimeric.98. in vivo assays using mice and cynomolgus monkeys and in clinical studies [113. and the anti-IL12/23 antibody ustekinumab (tradename Stelara®) for treatment of psoriasis [109].114]. This antibody was selected from the Vaughan library [112] against the B lymphocyte stimulator (BLyS). Here. part of AstraZeneca). Two lead scFvs were further optimised by randomisation of the CDR3 of the heavy chain and selection on BLyS by phage display. The anti-TNF-alpha antibody adalimumab (tradename Humira®) was isolated using antibody phage display by guided selection using a murine antibody as template [10.115].110. the first approved antibody muronomab-CD3 (tradename Orthoclone OKT3®) was directed against the T-cell marker CDR to minimise possible organ rejection after organ transplantations. 16 417 toxins [44] or cytokines like tumor necrosis factor (TNF) alpha [99]. Examples are infliximab (tradename Remicade®) against TNF-alpha for treatment of rheumatoid arthritis and other autoimmune diseases [103] or the anti-EGFR antibody cetuximab (tradename Erbitux®) for colorectal cancer treatment [104]. because the affinity of some antibodies is decreased after converting from the scFv to the IgG format [113. These human antibodies are derived from transgenic mice expressing the human antibody gene repertoire. the first fully human antibody was commercially available. several therapeutic antibodies derived from phage display are in clinical development [33.111]. an interleukin-1β (Il-1β)-specific antibody for treatment of cryopyrin-associated periodic syndromes [108]. mainly systemic lupus erythematosus (SLE). To date. The best scFvs were converted into the IgG format and neutralisation efficiency of the IgG were analysed again [113]. the initial binders were ranked by the efficiency to block BLyS binding to the BLyS receptor in cell based assays. One clone "LymphoStat-B" was further analysed in biochemical. constant domains of an antibody are human and the variable domains are murine. Other FDA-approved human antibodies are the anti-EGFR antibody panitumumab (tradename Vectibix®) [106] and golimumab (tradename Simponi®) [107] directed against TNF-alpha. Most phage display derived antibodes are generated by CAT (now MedImmune. This antibody was generated by CAT for treatment of autoimmune diseases. The advantage of antibody phage display is the direct isolation of human antibodies in comparison to antibodies generated by mouse hybridoma technology where a laborious humanisation of the lead candidates is necessary. Further examples are canakinumab (tradename Ilaris®). This step is important. blocking of receptors like epidermal growth factor receptor (EGFR) [100]. An example for the generation of human antibodies by phage display and further development is belimumab (tradename Benlysta®).Molecules 2011. Morphosys and Dyax. ScFv with an increased affinity were again converted into the IgG format and analysed regarding neutralisation In 1986. Here. This antibody is binding to the protective antigen (PA83) of Bacillus anthracis and was also developed by CAT [116]. A further phage display derived antibody which is currently under review by the FDA is raxibacumab. in vitro. Examples are trastuzumab (tradename Herceptin®) an anti-human epidermal growth factor receptor 2 (HER2) binder for treatment of breast cancer [105]. In 2002. When using transgenic mice comprising the human antibody repertoire human .

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