Recombinant Antibodies and In Vitro Selection Technologies

  • C. Ronald GeyerEmail author
  • John McCafferty
  • Stefan Dübel
  • Andrew R. M. Bradbury
  • Sachdev S. Sidhu
Part of the Methods in Molecular Biology book series (MIMB, volume 901)


Over the past decade, the accumulation of detailed knowledge of antibody structure and function has enabled antibody phage display to emerge as a powerful in vitro alternative to hybridoma methods for creating antibodies. Many antibodies produced using phage display technology have unique properties that are not obtainable using traditional hybridoma technologies. In phage display, selections are performed under controlled, in vitro conditions that are tailored to suit demands of the antigen and the sequence encoding the antibody is immediately available. These features obviate many of the limitations of hybridoma methodology, and because the entire process relies on scalable molecular biology techniques, phage display is also suitable for high-throughput applications. Thus, antibody phage display technology is well suited for genome-scale biotechnology and therapeutic applications. This review describes the antibody phage display technology and highlights examples of antibodies with unique properties that cannot easily be obtained by other technologies.

Key words

In vitro selection Phage display Antibodies 


  1. 1.
    von Behring E, Kitasato S (1890) Über das zustandekommen der diphtherie-immunität und der tetanus-immunität bei thieren. Deut Med Wochenzeitschr 16:1113–1114CrossRefGoogle Scholar
  2. 2.
    Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497PubMedCrossRefGoogle Scholar
  3. 3.
    Courtenay-Luck NS, Epenetos AA, Moore R et al (1986) Development of primary and secondary immune responses to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasms. Cancer Res 46:6489–6493PubMedGoogle Scholar
  4. 4.
    Tjandra JJ, Ramadi L, McKenzie IF (1990) Development of human anti-murine antibody (HAMA) response in patients. Immunol Cell Biol 68:367–376PubMedCrossRefGoogle Scholar
  5. 5.
    Almagro JC, Fransson J (2008) Humanization of antibodies. Front Biosci 13:1619–1633PubMedGoogle Scholar
  6. 6.
    Hwang WYK, Foote J (2005) Immunogenicity of engineered antibodies. Methods 36:3–10PubMedCrossRefGoogle Scholar
  7. 7.
    Kashmiri SVS, De Pascalis R, Gonzales NR, Schlom J (2005) SDR grafting—a new approach to antibody humanization. Methods 36:25–34PubMedCrossRefGoogle Scholar
  8. 8.
    Studnicka GM, Soares S, Better M et al (1994) Human engineered monoclonal antibodies retain full specific binding activity by preserving non-CDR complementarity-modulating residues. Protein Eng 7:805–814PubMedCrossRefGoogle Scholar
  9. 9.
    Osbourn J, Groves M, Vaughan T (2005) From rodent reagents to human therapeutics using antibody guided selection. Methods 36: 61–68PubMedCrossRefGoogle Scholar
  10. 10.
    Fishwild DM, O’Donnell SL, Bengoechea T et al (1996) High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat Biotechnol 14:845–851PubMedCrossRefGoogle Scholar
  11. 11.
    Jakobovits A (1995) Production of fully human antibodies by transgenic mice. Curr Opin Biotechnol 6:561–566PubMedCrossRefGoogle Scholar
  12. 12.
    Kuroiwa Y, Kasinathan P, Sathiyaseelan T et al (2009) Antigen specific human polyclonal antibodies from hyperimmunized cattle. Nat Biotechnol 27:173–181PubMedCrossRefGoogle Scholar
  13. 13.
    Lonberg N, Huszar D (1995) Human antibodies from transgenic mice. Int Rev Immunol 13:65–93PubMedCrossRefGoogle Scholar
  14. 14.
    Weiner LM (2006) Fully human therapeutic monoclonal antibodies. J Immunother 29:1–9PubMedCrossRefGoogle Scholar
  15. 15.
    Winter G, Milstein C (1991) Man-made antibodies. Nature 349:293–299PubMedCrossRefGoogle Scholar
  16. 16.
    McCafferty J, Griffiths AD, Winter G et al (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554PubMedCrossRefGoogle Scholar
  17. 17.
    Marks JD, Hoogenboom HR, Bonnert TP et al (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Bio 222:581–597CrossRefGoogle Scholar
  18. 18.
    Breitling F, Dübel S, Seehaus T (1991) A surface expression vector for antibody screening. Gene 104:147–153PubMedCrossRefGoogle Scholar
  19. 19.
    Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557PubMedCrossRefGoogle Scholar
  20. 20.
    Feldhaus MJ, Siegel RW, Opresko LK et al (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol 21:163–170PubMedCrossRefGoogle Scholar
  21. 21.
    He M, Taussig MJ (2007) Rapid discovery of protein interactions by cell-free protein technologies. Biochem Soc Trans 35:962–965PubMedCrossRefGoogle Scholar
  22. 22.
    Jostock T, Dübel S (2005) Screening of molecular repertoires by microbial surface display. Comb Chem High Throughput Screen 8:127–133PubMedCrossRefGoogle Scholar
  23. 23.
    Paschke M (2006) Phage display systems and their applications. Appl Microbiol Biotechnol 70:2–11PubMedCrossRefGoogle Scholar
  24. 24.
    Zahnd C, Amstutz P, Pluckthun A (2007) Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat Methods 4:269–279PubMedCrossRefGoogle Scholar
  25. 25.
    Chao G, Lau WL, Hackel BJ et al (2006) Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1: 755–768PubMedCrossRefGoogle Scholar
  26. 26.
    Benhar I (2007) Design of synthetic antibody libraries. Expert Opin Biol Ther 7:763–779PubMedCrossRefGoogle Scholar
  27. 27.
    Bradbury AR, Marks JD (2004) Antibodies from phage antibody libraries. J Immunol Methods 290:29–49PubMedCrossRefGoogle Scholar
  28. 28.
    Dübel S, Stoevesandt O, Taussig MJ et al (2010) Generating recombinant antibodies to the complete human proteome. Trends Biotechnol 28:333–339PubMedCrossRefGoogle Scholar
  29. 29.
    Mersmann M, Meier D, Mersmann J et al (2010) Towards proteome scale antibody selections using phage display. New Biotechnol 27:118–128CrossRefGoogle Scholar
  30. 30.
    Schofield DJ et al (2007) Application of phage display to high throughput antibody generation and characterization. Genome Biol 8:R254PubMedCrossRefGoogle Scholar
  31. 31.
    Colwill K, Gräslund S, Persson MA et al (2011) A roadmap to generate renewable protein binders to the human proteome. Nat Methods 8:551–561PubMedCrossRefGoogle Scholar
  32. 32.
    Hust M, Meyer T, Voedisch B et al (2011) A human scFv antibody generation pipeline for proteome research. J Biotechnol 152: 159–170PubMedCrossRefGoogle Scholar
  33. 33.
    Koide A, Bailey CW, Huang X et al (1998) The fibronectin type III domain as a scaffold for novel binding proteins. J Mol Biol 284: 1141–1151PubMedCrossRefGoogle Scholar
  34. 34.
    Angenendt P, Wilde J, Kijanka G et al (2004) Seeing better through a MIST: evaluation of monoclonal recombinant antibody fragments on microarrays. Anal Chem 76:2916–2921PubMedCrossRefGoogle Scholar
  35. 35.
    Philibert P, Stoessel A, Wang W (2007) A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm. BMC Biotechnol 7:1472–6750CrossRefGoogle Scholar
  36. 36.
    Parsons HL, Earnshaw JC, Wilton J et al (1996) Directing phage selections towards specific epitopes. Protein Eng 9:1043–1049PubMedCrossRefGoogle Scholar
  37. 37.
    Lassen KS, Bradbury AR, Rehfeld JF et al (2008) Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibody. Electrophoresis 29:2557–2564PubMedCrossRefGoogle Scholar
  38. 38.
    Kehoe JW, Velappan N, Walbol M et al (2006) Using phage display to select antibodies recognizing post-translational modifications independently of sequence context. Mol Cell Proteomics 5:2350–2363PubMedCrossRefGoogle Scholar
  39. 39.
    Hoffhines AJ, Damoc E, Bridges KG et al (2006) Detection and purification of tyrosine-sulfated proteins using a novel anti-sulfotyrosine monoclonal antibody. J Biol Chem 281:37877–37887PubMedCrossRefGoogle Scholar
  40. 40.
    Raza A, Garcia-Rodriguez C, Lou J et al (2005) Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A. J Mol Biol 351:158–169CrossRefGoogle Scholar
  41. 41.
    Lee CV, Liang WC, Dennis MS et al (2004) High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J Mol Biol 340: 1073–1093PubMedCrossRefGoogle Scholar
  42. 42.
    Hanes J, Schaffitzel C, Knappik A et al (2000) Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat Biotechnol 18: 1287–1292PubMedCrossRefGoogle Scholar
  43. 43.
    Schier R, McCall A, Adams GP et al (1996) Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J Mol Biol 263:551–567PubMedCrossRefGoogle Scholar
  44. 44.
    Yang WP, Green K, Pinz-Sweeney S et al (1995) CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J Mol Biol 254:392–403PubMedCrossRefGoogle Scholar
  45. 45.
    Boder ET, Midelfort KS, Wittrup KD (2000) Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci USA 97:10701–10705PubMedCrossRefGoogle Scholar
  46. 46.
    Foote J, Eisen HN (1995) Kinetic and affinity limits on antibodies produced during immune responses. Proc Natl Acad Sci USA 92: 1254–1256PubMedCrossRefGoogle Scholar
  47. 47.
    Foote J, Eisen HN (2000) Breaking the affinity ceiling for antibodies and T cell receptors. Proc Natl Acad Sci USA 97:10679–10681PubMedCrossRefGoogle Scholar
  48. 48.
    Batista FD, Neuberger MS (1998) Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. Immunity 8:751–759PubMedCrossRefGoogle Scholar
  49. 49.
    Sidhu SS (2005) Phage display in biotechnology and drug discovery. CRC Press, Baco Raton, FLCrossRefGoogle Scholar
  50. 50.
    Ponsel D, Neugebauer J, Ladetzki-Baehs K et al (2011) High affinity, developability and functional size: the holy grail of combinatorial antibody library generation. Molecules 16:3675–3700PubMedCrossRefGoogle Scholar
  51. 51.
    Azzazy HM, Highsmith WE Jr (2002) Phage display technology: clinical applications and recent innovations. Clin Biochem 35: 425–445PubMedCrossRefGoogle Scholar
  52. 52.
    Benhar I (2007) Design of synthetic antibody libraries. Expert Opin Biol Ther 7:763–779PubMedCrossRefGoogle Scholar
  53. 53.
    Burton DR, Barbas CF III, Persson MA et al (1991) A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc Natl Acad Sci USA 88:10134–10137PubMedCrossRefGoogle Scholar
  54. 54.
    Kramer RA, Marissen WE, Goudsmit J et al (2005) The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries. Eur J Immunol 35:2131–2145PubMedCrossRefGoogle Scholar
  55. 55.
    de Carvalho NC, Williamson RA, Parren PW et al (2002) Neutralizing human Fab fragments against measles virus recovered by phage display. J Virol 76:251–258CrossRefGoogle Scholar
  56. 56.
    Zebedee SL, Barbas CF III, Hom YL et al (1992) Human combinatorial antibody libraries to hepatitis B surface antigen. Proc Natl Acad Sci USA 89:3175–3179PubMedCrossRefGoogle Scholar
  57. 57.
    Cai X, Garen A (1995) Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries. Proc Natl Acad Sci USA 92:6537–6541PubMedCrossRefGoogle Scholar
  58. 58.
    Vaughan TJ, Williams AJ, Pritchard K et al (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14:309–314PubMedCrossRefGoogle Scholar
  59. 59.
    Lloyd C, Lowe D, Edwards B et al (2009) Modelling the human immune response: performance of a 1011 human antibody repertoire against a broad panel of therapeutically relevant antigens. Protein Eng Des Sel 22: 159–168PubMedCrossRefGoogle Scholar
  60. 60.
    Sheets MD, Amersdorfer P, Finnern R et al (1998) Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci USA 95:6157–6162PubMedCrossRefGoogle Scholar
  61. 61.
    De Haard HJ (2002) Construction of large naive Fab libraries. Methods Mol Biol 178: 87–100PubMedGoogle Scholar
  62. 62.
    Nissim A, Hoogenboom HR, Tomlinson IM et al (1994) Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents. EMBO J 13:692–698PubMedGoogle Scholar
  63. 63.
    de Kruif J, Boel E, Logtenberg T (1995) Selection and application of human single chain Fv antibody fragments from a semi-synthetic phage antibody display library with designed CDR3 regions. J Mol Biol 248: 97–105PubMedCrossRefGoogle Scholar
  64. 64.
    Griffiths AD, Williams SC, Hartley O et al (1994) Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J 13:3245–3260PubMedGoogle Scholar
  65. 65.
    Hoogenboom HR, Winter G (1992) By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol 227:381–388PubMedCrossRefGoogle Scholar
  66. 66.
    Pini A, Viti F, Santucci A et al (1998) Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel. J Biol Chem 273: 21769–21776PubMedCrossRefGoogle Scholar
  67. 67.
    Hoet RM, Cohen EH, Kent RB et al (2005) Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat Biotechnol 23:344–348PubMedCrossRefGoogle Scholar
  68. 68.
    Lee CV, Liang WC, Dennis MS et al (2004) High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J Mol Biol 340: 1073–1093PubMedCrossRefGoogle Scholar
  69. 69.
    Söderlind E, Strandberg L, Jirholt P et al (2000) Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat Biotechnol 18:852–856PubMedCrossRefGoogle Scholar
  70. 70.
    Rothe C, Urlinger S, Lohning C et al (2008) The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. J Mol Biol 376:1182–1200PubMedCrossRefGoogle Scholar
  71. 71.
    Sidhu SS, Li B, Chen Y, Fellouse FA et al (2004) Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J Mol Biol 338:299–310PubMedCrossRefGoogle Scholar
  72. 72.
    Fellouse FA, Wiesmann C, Sidhu SS (2004) Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc Natl Acad Sci USA 101:12467–12472PubMedCrossRefGoogle Scholar
  73. 73.
    Fellouse FA, Li B, Compaan DM, Peden AA et al (2005) Molecular recognition by a binary code. J Mol Biol 348:1153–1162PubMedCrossRefGoogle Scholar
  74. 74.
    Russel M, Linderoth NA, Sali A (1997) Filamentous phage assembly: variation on a protein export theme. Gene 192:23–32PubMedCrossRefGoogle Scholar
  75. 75.
    Iannolo G, Minenkova O, Petruzzelli R et al (1995) Modifying filamentous phage capsid: limits in the size of the major capsid protein. J Mol Biol 248:835–844PubMedCrossRefGoogle Scholar
  76. 76.
    Kretzschmarm T, Geiser M (1995) Evaluation of antibodies fused to minor coat protein III and major coat protein VIII of bacteriophage M13. Gene 155:61–65CrossRefGoogle Scholar
  77. 77.
    Rondot S, Koch J, Breitling F et al (2001) A helper phage to improve single chain antibody presentation in phage display. Nat Biotechnol 19:75–78PubMedCrossRefGoogle Scholar
  78. 78.
    Nelson AL, Dhimolea E, Reichert JM (2010) Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov 9:767–774PubMedCrossRefGoogle Scholar
  79. 79.
    Edwards BM, Barash SC, Main SH et al (2003) The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS. J Mol Biol 334:103–118PubMedCrossRefGoogle Scholar
  80. 80.
    Baker KP, Edwards BM, Main SH et al (2003) Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator. Arthritis Rheum 48:3253–3265PubMedCrossRefGoogle Scholar
  81. 81.
    Lloyd C, Lowe D, Edwards B et al (2009) Modelling the human immune response: performance of a 1011 human antibody repertoire against a broad panel of therapeutically relevant antigens. Protein Eng Des Sel 22:159–168PubMedCrossRefGoogle Scholar
  82. 82.
    Dong J, Demarest SJ, Sereno A et al (2010) Combination of two insulin-like growth factor-I receptor inhibitory antibodies targeting distinct epitopes leads to an enhanced antitumor response. Mol Cancer Ther 9:2593–2604PubMedCrossRefGoogle Scholar
  83. 83.
    Doern A, Cao X, Sereno A et al (2009) Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo. J Biol Chem 284:10254–10267PubMedCrossRefGoogle Scholar
  84. 84.
    Dong J, Sereno A, Snyder WB et al (2011) Stable IgG-like bispecific antibodies directed toward the type I insulin-like growth factor receptor demonstrate enhanced ligand blockade and anti-tumor activity. J Biol Chem 286:4703–4717PubMedCrossRefGoogle Scholar
  85. 85.
    Baselga J, Gelmon KA, Verma S et al (2010) Phase II trial of pertuzumab and trastuzumab in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer that progressed during prior trastuzumab therapy. J Clin Oncol 28:1138–1144PubMedCrossRefGoogle Scholar
  86. 86.
    Wu Y, Cain-Hom C, Choy L et al (2010) Therapeutic antibody targeting of individual Notch receptors. Nature 464:1052–1057PubMedCrossRefGoogle Scholar
  87. 87.
    Xie MH, Yuan J, Adams C et al (1997) Direct demonstration of MuSK involvement in acetylcholine receptor clustering through identification of agonist ScFv. Nat Biotechnol 15:768–771PubMedCrossRefGoogle Scholar
  88. 88.
    Ellmark P, Andersson H, Abayneh S et al (2008) Identification of a strongly activating human anti-CD40 antibody that suppresses HIV type 1 infection. AIDS Res Hum Retroviruses 24:367–373PubMedCrossRefGoogle Scholar
  89. 89.
    Dobson CL, Main S, Newton P et al (2009) Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism. MAbs 1:552–562PubMedCrossRefGoogle Scholar
  90. 90.
    Eisenhardt SU, Schwarz M, Bassler N et al (2007) Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules. Nat Protoc 2:3063–3073PubMedCrossRefGoogle Scholar
  91. 91.
    Huie MA, Cheung MC, Muench MO et al (2001) Antibodies to human fetal erythroid cells from a nonimmune phage antibody library. Proc Natl Acad Sci USA 98:2682–2687PubMedCrossRefGoogle Scholar
  92. 92.
    Noronha EJ, Wang X, Desai SA et al (1998) Limited diversity of human scFv fragments isolated by panning a synthetic phage-display scFv library with cultured human melanoma cells. J Immunol 161:2968–2976PubMedGoogle Scholar
  93. 93.
    Ridgway JB, Ng E, Kern JA et al (1999) Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and nontumor cell lines. Cancer Res 59:2718–2723PubMedGoogle Scholar
  94. 94.
    Van Ewijk W, de Kruif J, Germeraad WT et al (1997) Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments. Proc Natl Acad Sci USA 94:3903–3908PubMedCrossRefGoogle Scholar
  95. 95.
    Giordano RJ, Cardo-Vila M, Lahdenranta J et al (2001) Biopanning and rapid analysis of selective interactive ligands. Nat Med 7:1249–1253PubMedCrossRefGoogle Scholar
  96. 96.
    Williams BR, Sharon J (2002) Polyclonal anti-colorectal cancer Fab phage display library selected in one round using density gradient centrifugation to separate antigen-bound and free phage. Immunol Lett 81:141–148PubMedCrossRefGoogle Scholar
  97. 97.
    Osbourn JK, Derbyshire EJ, Vaughan TJ et al (1998) Pathfinder selection: in situ isolation of novel antibodies. Immunotechnology 3:293–302PubMedCrossRefGoogle Scholar
  98. 98.
    Osbourn JK, Earnshaw JC, Johnson KS et al (1998) Directed selection of MIP-1 alpha neutralizing CCR5 antibodies from a phage display human antibody library. Nat Biotechnol 16:778–781PubMedCrossRefGoogle Scholar
  99. 99.
    Poul MA, Becerril B, Nielsen UB et al (2000) Selection of tumor-specific internalizing human antibodies from phage libraries. J Mol Biol 301:1149–1161PubMedCrossRefGoogle Scholar
  100. 100.
    Zhou Y, Marks JD (2009) Identification of target and function specific antibodies for effective drug delivery. Methods Mol Biol 525:145–160PubMedCrossRefGoogle Scholar
  101. 101.
    Crépin R, Goenaga AL, Jullienne B et al (2010) Development of human single-chain antibodies to the transferrin receptor t, hat effectively antagonize the growth of leukemias and lymphomas. Cancer Res 70:5497–5506PubMedCrossRefGoogle Scholar
  102. 102.
    Park JW, Kirpotin DB, Hong K et al (2001) Tumor targeting using anti-her2 immunoliposomes. J Control Release 74:95–113PubMedCrossRefGoogle Scholar
  103. 103.
    Nielsen UB, Kirpotin DB, Pickering EM et al (2002) Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis. Biochim Biophys Acta 1591:109–118PubMedCrossRefGoogle Scholar
  104. 104.
    Velappan N, Martinez JS, Valero R et al (2007) Selection and characterization of scFv antibodies against the Sin Nombre hantavirus nucleocapsid protein. J Immunol Methods 321:60–69PubMedCrossRefGoogle Scholar
  105. 105.
    Cabezas S, Rojas G, Pavon A et al (2009) Phage-displayed antibody fragments recognizing dengue 3 and dengue 4 viruses as tools for viral serotyping in sera from infected individuals. Arch Virol 154:1035–1045PubMedCrossRefGoogle Scholar
  106. 106.
    Cabezas S, Rojas G, Pavon A et al (2008) Selection of phage-displayed human antibody fragments on Dengue virus particles captured by a monoclonal antibody: application to the four serotypes. J Virol Methods 147:235–243PubMedCrossRefGoogle Scholar
  107. 107.
    Lim AP, Chan CE, Wong SK et al (2008) Neutralizing human monoclonal antibody against H5N1 influenza HA selected from a Fab-phage display library. Virol J 5:130PubMedCrossRefGoogle Scholar
  108. 108.
    Okada J, Ohshima N, Kubota-Koketsu R et al (2010) Monoclonal antibodies in man that neutralized H3N2 influenza viruses were classified into three groups with distinct strain specificity: 1968–1973, 1977–1993 and 1997–2003. Virology 397:322–330PubMedCrossRefGoogle Scholar
  109. 109.
    Meissner F, Maruyama T, Frentsch M et al (2002) Detection of antibodies against the four subtypes of ebola virus in sera from any species using a novel antibody-phage indicator assay. Virology 300:236–243PubMedCrossRefGoogle Scholar
  110. 110.
    Kirsch MI, Hülseweh B, Nacke C et al (2008) Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV). BMC Biotechnol 8:66PubMedCrossRefGoogle Scholar
  111. 111.
    Hayhurst A, Happe S, Mabry R et al (2003) Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis. J Immunol Methods 276:185–196PubMedCrossRefGoogle Scholar
  112. 112.
    Zou N, Newsome T, Li B et al (2007) Human single-chain Fv antibodies against Burkholderia mallei and Burkholderia pseudomallei. Exp Biol Med (Maywood) 232:550–556Google Scholar
  113. 113.
    Maynard JA, Maassen CB, Leppla SH et al (2002) Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nat Biotechnol 20:597–601PubMedCrossRefGoogle Scholar
  114. 114.
    Wild MA, Xin H, Maruyama T et al (2003) Human antibodies from immunized donors are protective against anthrax toxin in vivo. Nat Biotechnol 21:1305–1306PubMedCrossRefGoogle Scholar
  115. 115.
    Steiniger SC, Altobell LJ 3rd, Zhou B, Janda KD (2007) Selection of human antibodies against cell surface-associated oligomeric anthrax protective antigen. Mol Immunol 44:2749–2755PubMedCrossRefGoogle Scholar
  116. 116.
    Pelat T, Hust M, Laffly E et al (2007) ­High-affinity, human antibody-like antibody ­fragment (single-chain variable fragment) neutralizing the lethal factor (LF) of Bacillus anthracis by inhibiting protective antigen-LF complex formation. Antimicrob Agents Chemother 51:2758–2764PubMedCrossRefGoogle Scholar
  117. 117.
    Cirino NM, Sblattero D, Allen D et al (1999) Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors. Infect Immun 67:2957–2963PubMedGoogle Scholar
  118. 118.
    Zhou B, Wirsching P, Janda KD (2002) Human antibodies against spores of the genus Bacillus: a model study for detection of and protection against anthrax and the bioterrorist threat. Proc Natl Acad Sci USA 99:5241–5246PubMedCrossRefGoogle Scholar
  119. 119.
    Sui J, Hwang WC, Perez S et al (2009) Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16:265–273PubMedCrossRefGoogle Scholar
  120. 120.
    Sun L, Lu X, Li C et al (2009) Generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza A H5N1 viruses. PLoS One 4:e5476PubMedCrossRefGoogle Scholar
  121. 121.
    Throsby M, van den Brink E, Jongeneelen M et al (2008) Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM  +  memory B cells. PLoS One 3:e3942PubMedCrossRefGoogle Scholar
  122. 122.
    Ayriss J, Woods T, Bradbury A, Pavlik P (2007) High-throughput screening of single-chain antibodies using multiplexed flow cytometry. J Proteome Res 6:1072–1082PubMedCrossRefGoogle Scholar
  123. 123.
    Pershad K, Pavlovic JD, Gräslund S et al (2010) Generating a panel of highly specific antibodies to 20 human SH2 domains by phage display. Protein Eng Des Sel 23:279–288PubMedCrossRefGoogle Scholar
  124. 124.
    Parsons HL, Earnshaw JC, Wilton J et al (1996) Directing phage selections towards specific epitopes. Protein Eng 9:1043–1049PubMedCrossRefGoogle Scholar
  125. 125.
    Mutuberria R, Satijn S, Huijbers A et al (2004) Isolation of human antibodies to tumor-associated endothelial cell markers by in vitro human endothelial cell selection with phage display libraries. J Immunol Methods 287:31–47PubMedCrossRefGoogle Scholar
  126. 126.
    Cohen CJ, Denkberg G, Lev A et al (2003) Recombinant antibodies with MHC-restricted, peptide-specific, T-cell receptor-like specificity: new tools to study antigen presentation and TCR-peptide-MHC interactions. J Mol Recognit 16:324–332PubMedCrossRefGoogle Scholar
  127. 127.
    Engberg J, Krogsgaard M, Fugger L (1999) Recombinant antibodies with the antigen-specific, MHC restricted specificity of T cells: novel reagents for basic and clinical investigations and immunotherapy. Immunotechnology 4:273–278PubMedCrossRefGoogle Scholar
  128. 128.
    Stryhn A, Andersen PS, Pedersen LO et al (1996) Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex. Proc Natl Acad Sci USA 93:10338–10342PubMedCrossRefGoogle Scholar
  129. 129.
    Villa A, Trachsel E, Kaspar M et al (2008) A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo. Int J Cancer 122:2405–2413PubMedCrossRefGoogle Scholar
  130. 130.
    Pini A, Viti F, Santucci A et al (1998) Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel. J Biol Chem 273:21769–21776PubMedCrossRefGoogle Scholar
  131. 131.
    Schliemann C, Neri D (2010) Antibody-based vascular tumor targeting. Recent Results Cancer Res 180:201–216PubMedCrossRefGoogle Scholar
  132. 132.
    Nizak C, Monier S, del Nery E et al (2003) Recombinant antibodies to the small GTPase Rab6 as conformation sensors. Science 300:984–987PubMedCrossRefGoogle Scholar
  133. 133.
    Gao J, Sidhu SS, Wells JA (2009) Two-state selection of conformation-specific antibodies. Proc Natl Acad Sci USA 106:3071–3076PubMedCrossRefGoogle Scholar
  134. 134.
    Eisenhardt SU, Schwarz M, Bassler N, Peter K (2007) Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules. Nat Protoc 2:3063–3073PubMedCrossRefGoogle Scholar
  135. 135.
    Rothlisberger D, Pos KM, Pluckthun A (2004) An antibody library for stabilizing and crystallizing membrane proteins - selecting binders to the citrate carrier CitS. FEBS Lett 564:340–348PubMedCrossRefGoogle Scholar
  136. 136.
    Uysal S, Vásquez V, Tereshko V et al (2009) Crystal structure of full-length KcsA in its closed conformation. Proc Natl Acad Sci USA 106:6644–6649PubMedCrossRefGoogle Scholar
  137. 137.
    Ye JD, Tereshko V, Frederiksen JK et al (2008) Synthetic antibodies for specific recognition and crystallization of structured RNA. Proc Natl Acad Sci USA 105:82–87PubMedCrossRefGoogle Scholar
  138. 138.
    Koldobskaya Y, Duguid EM, Shechner DM et al (2010) A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination. Nat Struct Mol Biol 18:100–106PubMedCrossRefGoogle Scholar
  139. 139.
    Monigatti F, Gasteiger E, Bairoch A, Jung E (2002) The Sulfinator: predicting tyrosine sulfation sites in protein sequences. Bioinformatics 18:769–770PubMedCrossRefGoogle Scholar
  140. 140.
    Kehoe JW, Velappan N, Walbolt M et al (2006) Using phage display to select antibodies recognizing post-translational modifications independently of sequence context. Mol Cell Proteomics 5:2350–2363PubMedCrossRefGoogle Scholar
  141. 141.
    Thie H, Voedisch B, Dübel S et al (2009) Affinity maturation by phage display. Methods Mol Biol 525:309–322PubMedCrossRefGoogle Scholar
  142. 142.
    Cadwell RC, Joyce GF (1994) Mutagenic PCR. PCR Methods Appl 3:S136–S140PubMedGoogle Scholar
  143. 143.
    Stemmer WP (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91:10747–10751PubMedCrossRefGoogle Scholar
  144. 144.
    Fellouse FA, Wiesmann C, Sidhu SS (2004) Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc Natl Acad Sci USA 101:12467–12472PubMedCrossRefGoogle Scholar
  145. 145.
    Liang WC, Wu X, Peale FV et al (2006) Cross-species vascular endothelial growth factor (VEGF)-blocking antibodies completely inhibit the growth of human tumor xenografts and measure the contribution of stromal VEGF. J Biol Chem 281:951–961PubMedCrossRefGoogle Scholar
  146. 146.
    Lee CV, Hymowitz SG, Wallweber HJ et al (2006) Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells. Blood 108:3103–3111PubMedCrossRefGoogle Scholar
  147. 147.
    Fagète S, Ravn U, Gueneau F et al (2009) Specificity tuning of antibody fragments to neutralize two human chemokines with a single agent. MAbs 1:288–296PubMedCrossRefGoogle Scholar
  148. 148.
    Bostrom J, Yu SF, Kan D et al (2009) Variants of the antibody herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323:1610–1614PubMedCrossRefGoogle Scholar
  149. 149.
    Volk WA, Bizzini B, Snyder RM et al (1984) Neutralization of tetanus toxin by distinct monoclonal antibodies binding to multiple epitopes on the toxin molecule. Infect Immun 45:604–609PubMedGoogle Scholar
  150. 150.
    Zwick MB, Labrijn AF, Wang M et al (2001) Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J Virol 75:10892–10905PubMedCrossRefGoogle Scholar
  151. 151.
    Cheson BD, Leonard JP (2008) Monoclonal antibody therapy for B-cell non-Hodgkin’s lymphoma. N Eng J Med 359:613–626CrossRefGoogle Scholar
  152. 152.
    de Kruif J, Logtenberg T (1996) Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library. J Biol Chem 271: 7630–7634PubMedCrossRefGoogle Scholar
  153. 153.
    Thie H, Binius S, Schirrmann T et al (2009) Multimerization domains for antibody phage display and antibody production. New Biotechnol 26:314–321CrossRefGoogle Scholar
  154. 154.
    Hudson PJ, Kortt AA (1999) High avidity scFv multimers; diabodies and triabodies. J Immunol Methods 231:177–189PubMedCrossRefGoogle Scholar
  155. 155.
    Dübel S, Breitling F, Kontermann R et al (1995) Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv). J Immunol Methods 178:201–209PubMedCrossRefGoogle Scholar
  156. 156.
    Huang D, Shusta EV (2006) A yeast platform for the production of single-chain antibody-green fluorescent protein fusions. Appl Environ Microbiol 72:7748–7759PubMedCrossRefGoogle Scholar
  157. 157.
    Hink MA, Griep RA, Borst JW et al (2000) Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein. J Biol Chem 275:17556–17560PubMedCrossRefGoogle Scholar
  158. 158.
    Casey JL, Coley AM, Tilley LM et al (2000) Green fluorescent antibodies: novel in vitro tools. Protein Eng 13:445–452PubMedCrossRefGoogle Scholar
  159. 159.
    Griep RA, van Twisk C, Kerschbaumer RJ et al (1999) pSKAP/S: an expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins. Protein Expr Purif 16:63–69PubMedCrossRefGoogle Scholar
  160. 160.
    Al-Mrabeh A, Ziegler A, Cowan G et al (2009) A fully recombinant ELISA using in vivo biotinyla.ted antibody fragments for the detection of potato leafroll virus. J Virol Methods 159:200–205PubMedCrossRefGoogle Scholar
  161. 161.
    Predonzani A, Arnoldi F, Lopez-Requena A et al (2008) In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system. BMC Biotechnol 8:41PubMedCrossRefGoogle Scholar
  162. 162.
    Warren DJ, Bjerner J, Paus E et al (2005) Use of an in vivo biotinylated single-chain antibody as capture reagent in an immunometric assay to decrease the incidence of interference from heterophilic antibodies. Clin Chem 51: 830–838PubMedCrossRefGoogle Scholar
  163. 163.
    Cloutier SM, Couty S, Terskikh A et al (2000) Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin. Mol Immunol 37:1067–1077PubMedCrossRefGoogle Scholar
  164. 164.
    Moutel S et al (2009) A multi-Fc-species system for recombinant antibody production. BMC Biotechnol 9:14PubMedCrossRefGoogle Scholar
  165. 165.
    Hu S, Shively L, Raubitschek A et al (1996) Minibody: a novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts. Cancer Res 56:3055–3061PubMedGoogle Scholar
  166. 166.
    Gilliland LK, Norris NA, Marquardt H et al (1996) Rapid and reliable cloning of antibody variable regions and generation of recombinant single chain antibody fragments. Tissue Antigens 47:1–20PubMedCrossRefGoogle Scholar
  167. 167.
    Shan D, Press OW, Tsu TT et al (1999) Characterization of scFv-Ig constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths. J Immunol 162:6589–6595PubMedGoogle Scholar
  168. 168.
    Kontermann RE (2010) Alternative antibody formats. Curr Opin Mol Ther 12:176–183PubMedGoogle Scholar
  169. 169.
    Merchant AM, Zhu Z, Yuan JQ et al (1998) An efficient route to human bispecific IgG. Nat Biotechnol 16:677–681PubMedCrossRefGoogle Scholar
  170. 170.
    Ridgway JB, Presta LG et al (1996) ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9:617–621PubMedCrossRefGoogle Scholar
  171. 171.
    Coloma MJ, Morrison SL (1997) Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol 15:159–163PubMedCrossRefGoogle Scholar
  172. 172.
    Dong J, Sereno A, Snyder WB et al (2011) Stable IgG-like bispecific antibodies directed toward the type I insulin-like growth factor receptor demonstrate enhanced ligand blockade and anti-tumor activity. J Biol Chem 286:4703–4717PubMedCrossRefGoogle Scholar
  173. 173.
    Kortt AA, Dolezal O, Power BE et al (2001) Dimeric and trimeric antibodies: high avidity scFvs for cancer targeting. Biomol Eng 18: 95–108PubMedCrossRefGoogle Scholar
  174. 174.
    Lawrence LJ, Kortt AA, Iliades P et al (1998) Orientation of antigen binding sites in dimeric and trimeric single chain Fv antibody fragments. FEBS Lett 425:479–484PubMedCrossRefGoogle Scholar
  175. 175.
    Perisic O, Webb PA, Holliger P et al (1994) Crystal structure of a diabody, a bivalent antibody fragment. Structure 2:1217–1226PubMedCrossRefGoogle Scholar
  176. 176.
    Atwell JL, Breheney KA, Lawrence LJ et al (1999) scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies. Protein Eng 12: 597–604PubMedCrossRefGoogle Scholar
  177. 177.
    Pei XY, Holliger P, Murzin AG et al (1997) The 2.0-A resolution crystal structure of a trimeric antibody fragment with noncognate VH-VL domain pairs shows a rearrangement of VH CDR3. Proc Natl Acad Sci USA 94:9637–9642PubMedCrossRefGoogle Scholar
  178. 178.
    Le Gall F, Kipriyanov SM, Moldenhauer G et al (1999) Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Lett 453:164–168PubMedCrossRefGoogle Scholar
  179. 179.
    Muller D, Kontermann RE (2010) Bispecific antibodies for cancer immunotherapy: current perspectives. BioDrugs 24:89–98PubMedCrossRefGoogle Scholar
  180. 180.
    Beck A, Wagner-Rousset E, Bussat MC et al (2008) Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. Curr Pharm Biotechnol 9:482–501PubMedCrossRefGoogle Scholar
  181. 181.
    Presta LG (2008) Molecular engineering and design of therapeutic antibodies. Curr Opin Immunol 20:460–470PubMedCrossRefGoogle Scholar
  182. 182.
    Hallborn J, Carlsson R (2002) Automated screening procedure for high-throughput generation of antibody fragments. Biotechniques Suppl, 30–37Google Scholar
  183. 183.
    Lou J, Marzari R, Verzillo V et al (2001) Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries. J Immunol Methods 253:233–242PubMedCrossRefGoogle Scholar
  184. 184.
    Kawe M, Forrer P, Amstutz P et al (2006) Isolation of intracellular proteinase inhibitors derived from designed ankyrin repeat proteins by genetic screening. J Biol Chem 281: 40252–40263PubMedCrossRefGoogle Scholar
  185. 185.
    Glanville J, Zhai W, Berka J et al (2009) Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire. Proc Natl Acad Sci USA 106:20216–20221PubMedCrossRefGoogle Scholar
  186. 186.
    Ravn U, Gueneau F, Baerlocher L et al (2010) By-passing in vitro screening-next generation sequencing technologies applied to antibody display and in silico candidate selection. Nucleic Acids Res 38:e193PubMedCrossRefGoogle Scholar
  187. 187.
    Ge X, Mazor Y, Hunicke-Smith SP et al (2010) Rapid construction and characterization of synthetic antibody libraries without DNA amplification. Biotechnol Bioeng 106: 347–357PubMedGoogle Scholar
  188. 188.
    Fischer N (2011) Sequencing antibody repertoires: the next generation. MAbs 3: 17–20PubMedCrossRefGoogle Scholar
  189. 189.
    Reddy ST, Ge X, Miklos AE et al (2010) Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. Nat Biotechnol 28: 965–969PubMedCrossRefGoogle Scholar
  190. 190.
    Zhang H, Torkamani A, Jones TM et al (2011) Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems. Proc Natl Acad Sci USA 108: 13456–13461PubMedCrossRefGoogle Scholar
  191. 191.
    Bordeaux J, Welsh A, Agarwal S et al (2010) Antibody validation. Biotechniques 48: 197–209PubMedCrossRefGoogle Scholar
  192. 192.
    Pozner-Moulis S, Cregger M, Camp RL et al (2007) Antibody validation by quantitative analysis of protein expression using expression of Met in breast cancer as a model. Lab Invest 87:251–260PubMedCrossRefGoogle Scholar
  193. 193.
    Grimsey NL, Goodfellow CE, Scotter EL et al (2008) Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal! J Neurosci Methods 171:78–86PubMedCrossRefGoogle Scholar
  194. 194.
    Saper CB (2005) An open letter to our readers on the use of antibodies. J Comp Neurol 493:477–478PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • C. Ronald Geyer
    • 1
    Email author
  • John McCafferty
    • 2
  • Stefan Dübel
    • 3
  • Andrew R. M. Bradbury
    • 4
  • Sachdev S. Sidhu
    • 5
  1. 1.University of SaskatchewanSaskatoonCanada
  2. 2.University of CambridgeCambridgeUK
  3. 3.Technische Universität BraunschweigBraunschweigGermany
  4. 4.Los Alamos National LaboratoryLos AlamosUSA
  5. 5.University of TorontoTorontoCanada

Personalised recommendations