Hyperphage - a helperphage with improved panning efficiency in phage display


An Effective Tool for the Isolation of Recombinant Antibodies, Proteins or Peptides from Hyperphage-Packed Libraries.


A helper phage technology ("Hyperphage System") was developed by Rondot et al. (Nature Biotechnology 19:75-81, 2001). The Hyperphage System allows to improve antibody presentation in phage display by increasing the number of antibodies displayed per phage particle (up to 5 vs. 0.01) and thereby the system offers great advantage in the fields of functional gene analysis and proteomics. Panning of phages can be performed with small amounts of antigen and higher efficiency. For example, the application of universal libraries for antibody isolation can be improved by employing panning of hyperphage-packed libraries on blots of protein spots after 2-dimensional gel electrophoresis.

More Applications

Large numbers of open reading frames (ORFs) can be analyzed by panning against synthetic membrane- bound peptide epitopes. In cancer research, the hyperphage-packed library could be a tool to discover new tumour markers by panning against cellular surfaces.

The Hyperphage System

Hyperphages carry a deletion in the pIII gene. They are generated by an E. coli packaging cell line producing functional pIII which is used to package a phage genome with a pIII deletion. The resulting hyperphages carry functional pIII on their surface but lack the pIII gene in their genome. These hyperphages can then be used to infect bacteria with a phagemid library. Each of the resulting display phages carries several copies of the antibody or peptide on its surface, thus dramatically increasing panning efficiency.

Example with pSEX Phagemid Antibody Gene Library

Antigen binding was enhanced by more than two orders of magnitude by using hyperphage. Further, since the antibody carrying plasmid (phagemid) encodes a protease cleavage site between pIII and scFv fragment, the hyperphage-packed library can be eluted by protease treatment, allowing to elute the highest affinity binders, plus restoring wild-type infectivity phenotype to optimize the recovery of the antibody gene of interest.


  • increases panning efficiency
  • allows panning with reduced amount of panning antigen
  • identifies high and low affinity binders




  • Hust M., Frenzel, A., Tomszak, F, Kügler, J & Dübel, S. (2014) Antibody Phage Display. In: Dübel, S. & Reichert, J.M. (eds.) Handbook of Therapeutic Antibodies, 2nd ed. Wiley-VCH, Weinheim, ISBN 978-3-527-32937-3 p. 43-76
  • Hust, M., Frenzel, A., Schirmann, T. and Dübel, S. (2014) Selection of recombinant antibodies from antibody gene libraries. Methods Mol Biol. 1101, 305-320.2
  • Hust, M., Frenzel, A., Meyer, T., Schirmann, T. and Dübel, S. (2012). Construction of human naive antibody gene libraries. In: Antibody Engineering: Methods and Protocols, Ed: Chames, P., Meth Mol Biol 907, 85-107
  • Breitling, F., Broders, O., Helmsing, S., Hust, M & Dübel, S (2010) Improving phage display throughput by using Hyperphage, miniaturised titration and pVIII (g8p) ELISA. In: Antibody Engineering (2nd ed) Springer Protocols. Vol. 1, ISBN 978-3-642-01143-6, p. 197-206
  • Broders, O., Breitling, F., and Dübel, S. (2003). Hyperphage - Improving Antibody Presentation in Phage Display. Methods Mol Biol 205, 295-302.


Original papers on the successful use of the hyperphage system

  • Goffin L, Fagagnini S, Vicari A, Mamie C, Melhem H, Weder B, Lutz C, Lang S, Scharl M, Rogler G, Chvatchko Y, Hausmann M.Anti-MMP-9 Antibody: A Promising Therapeutic Strategy for Treatment of Inflammatory Bowel Disease Complications with Fibrosis.Inflamm Bowel Dis. 2016 Sep;22(9):2041-57. doi: 10.1097/MIB.0000000000000863
  • Mendonça M, Moreira GMSG, Conceição FR, Hust M, Mendonça KS, Moreira ÂN, et al. (2016) Fructose 1,6-Bisphosphate Aldolase, a Novel Immunogenic Surface Protein on Listeria Species. PLoS ONE 11(8): e0160544. doi:10.1371/journal.pone.0160544
  • Zantow, J., Just. S., Lagkouvardos, I.,  Kisling. S., Dübel, S., Lepage. P., Clave, T. & Hust, M. (2016) Mining gut microbiome oligopeptides by functional metaproteome display. Sci. Rep. 6:34337, DOI:10.1038/srep34337
  • Connor DO, Zantow J, Hust M, Bier FF, von Nickisch-Rosenegk M. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display. PLoS One. 2016 Feb 9;11(2):e0148986. doi: 10.1371/journal.pone.0148986 114.
  • Derman, Y., Selby, K., Miethe, S., Frenzel, A., Liu, Y., Rasetti-Escargueil, C., Avril, A., Pelat, T., Urbain, R., Fontayne, A., Thullier, P., Sesardic, D., Lindström, M., Hust, M. and Korkeala, H. (2016). Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody. Toxins 8: 25
  • Rohrbeck, A., Fühner, V., Schröder, A., Hagemann, S., Vu, X.-H., Berndt, S., Hust, M., Pich, A. and Just, I. (2016). Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody. Toxins 8:100
  • Miethe, S., Mazuet, C., Liu, Y., Tierney, R., Rasetti-Escargueil, C., Avril, A., Frenzel, A., Thullier, P., Pelat, T., Urbain, R., Fontayne, A., Sesardic, D., Hust, M. and Popoff, M.-R. (2016). Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B. PLoS ONE 11: e0161446
  • Blokzijl, A.,  Zieba, A.,  Hust, M.,  Schirrmann, T.,  Helmsing, S.,  Grannas, K.,  Hertz, E.,  Moren, A.,  Chen, L.,  Soderberg,, O., Moustakas, A., Dübel, S., & Landegren, U. (2016) Single chain antibodies as tools to study TGF-β regulated SMAD proteins in proximity ligation-based pharmacological screens. Mol Cell Proteomics 15, 1848-1856
  • Kibat, J, Schirrmann. T., Knape, M.J.,, Helmsing, S., Meier, D., Hust, M., Schröder, C., Bertinetti, D., Pardes, K., Funk, M., Lages, A., Giese, N., Herberg, F. W., Dübel, S. & Hoheisel, J.D.  (2016) Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality. N. Biotech. 33, 574-581
  • Rasetti-Escargueil, C., Avril, A., Tierney, R., Bak, N., Miethe, S., Chahboun, S., Mazuet, C., Popoff, M., Thullier, P., Hust, M., Pelat, T. and Sesardic, D. (2015). Development of human-like scFv-Fc neutralising Botulinum toxin type B. mAbs 6: 1161-1177
  • Miethe, S., Rasetti-Escargueil, C., Avril, A., Liu, Y., Chahboun, S., Korkeala, H., Mazuet, C., Popoff, M.-R., Pelat, T., Thullier. P., Sesardic, D. and Hust, M. (2015).  Development of human-like scFv-Fc neutralizing botulinum neurotoxin E. PLoS ONE 10: e0139905
  • Becker, M., Felsberger, A., Frenzel, A., Shattuck, W.M.C, Dyer, M., Kügler, J., Zantow, J., Mather, T. and Hust, M. (2015). Application of M13 Phage Display for Identifying Immunogenic Proteins from Tick (Ixodes scapularis) Saliva. BMC Biotechnology 15: 43
  • Avril, A., Miethe, S., Popoff, M., Mazuet, C., Chahboun, S., Rassetti-Escargueil, C., Sesardic, D., Thullier, P., Hust, M. and Pelat, T. (2015). Isolation of nanomolar scFvs of non-human primate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain. BMC Biotechnology 15: 86
  • Droste P., Frenzel A., Steinwand, M., Pelat, T., Thullier, P., Hust, M., Lashuel, H. and Dübel, S. (2015) Structural differences of amyloid-β fibrils revealed by antibodies from phage display. BMC Biotechnol.15:57
  • Zehner, M., Marschall, A.L.J., Erik Bos, E., Schloetel, J.-G., Kreer, C., Fehrenschild, D., Limmer, A., Ossendorp, F., Lang, T., Koster, A.J., Dübel, S., Burgdorf, S. (2015) Endosomal Sec61 mediates antigen translocation in the cytosol for cross-presentation. Immunity 42:850-863
  • Kügler, J., Wilke. S., Doris, M., Tomszak, F., Frenzel, A., Schirrmann, T., Dübel, S., Garritsen, H., Hock. B., Toleikis, L., Schütte, M. and Hust. M. (2015) Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnol. 15:10
  • Fuchs, M.,  Kämpfer, S., Spallek, R., Oehlmann, W., Prilop, W., Frank, R., Dübel, S., Singh, M. & Hust, M. (2014) Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B. BMC Biotechnol. 14:68
  • Hülseweh, B., Rülker, T., Pelat, T., Langermann, C., Frenzel, A., Schirrmann, T., Dübel, S., Thullier, P. & Hust, M., (2014) Human-like antibodies neutralizing Western Equine Encephalitis Virus. mAbs 6, 717 - 726
  • Trott, M., Weiß, S., Antoni, S., Koch, J., von Briesen, H., Hust, M. and Dietrich, U. (2014).  Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody. PLoS ONE 9:  e97478
  • Miethe, S. , Pelat, T., Avril, A., Popoff, M., Molgo, J., Rasetti Escargueil, C., Fontayne, A., Korkeala, H., Thullier, P., Sesardic, D. and Hust, M. (2014). Development of recombinant antibodies for biodefense using phage display. STO Scientific Publications, STO-MP-HFM239-16
  • Miethe, S., Rasetti-Escargueil, C., Liu, Y., Chahboun, S., Pelat, T., Avril, A., Frenzel, A., Schirrmann, T., Thullier, P., Sesardic, D. and Hust, M. (2014). Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library. mAbs 6: 446-459
  • Schirrmann, T. Frenzel, A., Linden, L., Stelte-Ludwig, B., Willuda, J., Harrenga, A., Dübel, S., Müller-Tiemann, B. & Trautwein, M. (2014) Evaluation of human pancreatic RNase as effector molecule in a therapeutic antibody platform. mAbs, J6(2)
  • Steinwand, M., Droste, P., Frenzel, A., Hust, M., Dübel, S. and Schirrmann, T. (2013) The influence of antibody fragment format on phage display based affinity maturation of IgG. mAbs, 6, 204-218  
  • Zhou, M., Meyer, T., Koch, S., Koch, J., Brill, B., von Briesen, H., Benito, J.M., Soriano, V., Haberl, A., Bickel, M., Dübel, S., Hust, M., and Dietrich, U. (2013) Identification of an epitope in the membrane proximal external region of gp41 targeted by neutralizing antibodies in plasma from an elite controller using an Env-tailored phage display library. Eur J immunol, 43, 499-509
  • Meyer, T.,  Schirrmann, T., Frenzel, A., Miethe, S., Stratmann-Selke, J., Gerlach, G.F., Strutzberg-Minder, K., Dübel, S., and Hust, M. (2012) Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display. BMC Biotechnol, 12:29
  • Wezler, X., Hust, M., Helmsing, S., Schirrmann, T. & Dübel, S. (2012) Human antibodies targeting CD30+ lymphomas. Hum. Antibod. 21:13-28
  • Rülker, T., Voß, L., Thullier, P., O’ Brien, L.M., Pelat, T., Perkins, S.D., Langermann, C., Schirrmann, T., Dübel, S., Marschall, H.-J., Hust, M. & Hülseweh, B. (2012) Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo. PLoS ONE, 7(5): e37242. doi:10.1371/journal.pone.0037242
  • Zhang, C., Helmsing, S., Schirrmann, T., Marschall, A.L.J., Zagrebelsky, M., Hust, M., Korte, M & Dübel, S. (2012) Suppression of p75 neurotrophin receptor surface expression with intrabodies influences Bcl-xL mRNA expression and neurite outgrowth in PC12 cells. PLoS ONE 7(1): e30684. doi:10.1371/journal.pone.0030684
  • Colwill, K., Renewable Protein Binder Working Group & Gräslund, S., (2011) A roadmap to generate renewable protein binders to the human proteome. Nature Meth. 8, 551–558
  • Hust, M., Meyer, T., Voedisch, B., Rülker, T., Thie, H., Schütte, M., Helmsing, S., Meier, D., Schirrmann, T., Dübel, S. (2011) A human scFv antibody generation pipeline for proteome research, J. Biotechnol. 152,159-170
  • Meyer, T., Stratmann-Selke, J., Meens, J., Schirrmann, T., Gerlach, G.F., Frank, R., Dübel, S., Strutzberg-Minder, K., Hust, M. (2010) Isolation of OmpD specific scFv fragments for the diagnosis of Salmonella Typhimurium. Vet. Microbiol. 147, 162-169
  • Mersmann, M., Meier, D., Mersmann, J., Helmsing, S., Nilsson, P., Gräslund, S., Structural Genomics Consortium,  Colwill, K., Hust M., and Dübel, S. (2010) Towards Proteome Scale Antibody Selections using Phage Display. N. Biotec, 27, 118-128
  • Schütte, M., Thullier, P., Pelat, T., Wezler, X., Rosenstock, P.,  Dominik, Hinz, D., Kirsch, M.I., Hasenberg, M.,   Frank, R., Schirrmann, T., Gunzer, M., Hust, M. & Dübel, S. (2009) Identification of a putative Crf splice variant and generation of  recombinant antibodies for the specific detection of Aspergillus  fumigatus (2009) PLoS ONE 4:e6625
  • Pelat, T., Hust, M., Hale, M., Lefranc, M.P., Dübel, S. & Thullier, P. (2009) Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity. BMC Biotechnol., 9:60
  • Naseem, S., Meens, J., Jores, J., Heller, M., Dübel, S., Hust, M. and Gerlach, G.F.. (2010) Phage display-based identification and potential diagnostic application of novel antigens from Mycoplasma mycoides subsp. mycoides small colony type. Veterinary Microbiology 142, 285-292
  • Kirsch, M.I., Hülseweh, B., Nacke, C., Rülker, T., Schirrmann, T., Marschall, H.-J., Hust, M. and Dübel, S. (2008). Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV).  BMC-Biotechnol. 8, 66
  • Kügler, J., Nieswandt, S., Gerlach, G.F., Meens, J., Schirrmann, T. and Hust, M. (2008). Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display. Applied Microbiology and Biotechnology 80, 447- 458
  • Thie H, Schirrmann T, Paschke M, Dübel S, Hust M. (2008) SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli. N Biotechnol. 5, 49-54
  • Pelat, T., Hust, M, Laffly, E., Condemine, F., Bottex, C., Vidal, D., Lefranc, M.P., Dübel, S., Thullier, P. (2007) A high affinity, human like antibody fragment (scFv) neutralising lethal factor (LF) of Bacillus anthracis by inhibiting the PA-LF complex formation. Antimicrob agents and chemother 51, 2758-2764
  • Soltes G, Hust M, Ng KKY, Bansal A, Field J, Stewart DIH, Dübel S, Cha S, Wiersma E (2007) On the influence of vector design on antibody phage display. J Biotech 127, 626-637
  • Hust, M., Meysing, M., Schirrmann, T., Selke, M., Meens, J., Gerlach, G.F. & Dübel, S. (2006) Enrichment of open reading frames presented on bacteriophage M13 using Hyperphage. Bio/Techniques, 41, 335-342
  • Kirsch, M., Zaman, M., Meier, D., Dübel, S. and Hust, M. (2005) Parameters affecting the display of antibodies on phage. J immunol Meth 301, 173-85
  • Rondot, S., Koch, J., Breitling, F., and Dübel, S. (2001). A helperphage to improve single chain antibody presentation in phage display. Nature Biotechnol. 19, 75-78