Beyond natural antibodies: the power of in vitro display technologies

Article metrics

Abstract

In vitro display technologies, best exemplified by phage and yeast display, were first described for the selection of antibodies some 20 years ago. Since then, many antibodies have been selected and improved upon using these methods. Although it is not widely recognized, many of the antibodies derived using in vitro display methods have properties that would be extremely difficult, if not impossible, to obtain by immunizing animals. The first antibodies derived using in vitro display methods are now in the clinic, with many more waiting in the wings. Unlike immunization, in vitro display permits the use of defined selection conditions and provides immediate availability of the sequence encoding the antibody. The amenability of in vitro display to high-throughput applications broadens the prospects for their wider use in basic and applied research.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The unique capabilities of in vitro selection offer advantages over the immunization of animals for antibody generation.
Figure 2: In vitro selected antibodies can recognize minute differences in small molecules.
Figure 3: Mechanisms for blocking or activating receptor signaling using antibodies.
Figure 4: An engineered dual specificity synthetic Fab.

Accession codes

Accessions

Protein Data Bank

References

  1. 1

    Love, J.C., Ronan, J.L., Grotenbreg, G.M., van der Veen, A.G. & Ploegh, H.L. A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat. Biotechnol. 24, 703–707 (2006).

  2. 2

    Jin, A. et al. A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood. Nat. Med. 15, 1088–1092 (2009).

  3. 3

    Reddy, S.T. et al. Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. Nat. Biotechnol. 28, 965–969 (2010).

  4. 4

    Smith, G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317 (1985).

  5. 5

    Scott, J.K. & Smith, G.P. Searching for peptide ligands with an epitope library. Science 249, 386–390 (1990).

  6. 6

    Skerra, A. & Pluckthun, A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038–1041 (1988).

  7. 7

    Larrick, J.W. et al. Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction. Biochem. Biophys. Res. Commun. 160, 1250–1256 (1989).

  8. 8

    Marks, J.D. et al. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597 (1991).

  9. 9

    Orlandi, R., Gussow, D.H., Jones, P.T. & Winter, G. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 3833–3837 (1989).

  10. 10

    Huse, W.D. et al. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246, 1275–1281 (1989).

  11. 11

    Sastry, L. et al. Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library. Proc. Natl. Acad. Sci. USA 86, 5728–5732 (1989).

  12. 12

    McCafferty, J., Griffiths, A.D., Winter, G. & Chiswell, D.J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554 (1990).

  13. 13

    Breitling, F., Dübel, S., Seehaus, T., Klewinghaus, I. & Little, M. A surface expression vector for antibody screening. Gene 104, 147–153 (1991).

  14. 14

    Boder, E.T. & Wittrup, K.D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

  15. 15

    Feldhaus, M.J. et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat. Biotechnol. 21, 163–170 (2003).

  16. 16

    Bordeaux, J. et al. Antibody validation. Biotechniques 48, 197–209 (2010).

  17. 17

    Jositsch, G. et al. Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice. Naunyn Schmiedebergs Arch. Pharmacol. 379, 389–395 (2009).

  18. 18

    Jensen, B.C., Swigart, P.M. & Simpson, P.C. Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific. Naunyn Schmiedebergs Arch. Pharmacol. 379, 409–412 (2009).

  19. 19

    Spicer, S.S., Spivey, M.A., Ito, M. & Schulte, B.A. Some ascites monoclonal antibody preparations contain contaminants that bind to selected Golgi zones or mast cells. J. Histochem. Cytochem. 42, 213–221 (1994).

  20. 20

    Pozner-Moulis, S., Cregger, M., Camp, R.L. & Rimm, D.L. Antibody validation by quantitative analysis of protein expression using expression of Met in breast cancer as a model. Lab. Invest. 87, 251–260 (2007).

  21. 21

    Grimsey, N.L. et al. Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal! J. Neurosci. Methods 171, 78–86 (2008).

  22. 22

    Saper, C.B. An open letter to our readers on the use of antibodies. J. Comp. Neurol. 493, 477–478 (2005).

  23. 23

    Paschke, M. Phage display systems and their applications. Appl. Microbiol. Biotechnol. 70, 2–11 (2006).

  24. 24

    Zahnd, C., Amstutz, P. & Pluckthun, A. Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat. Methods 4, 269–279 (2007).

  25. 25

    Chao, G. et al. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1, 755–768 (2006).

  26. 26

    Binz, H.K., Amstutz, P. & Pluckthun, A. Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23, 1257–1268 (2005).

  27. 27

    Binz, H.K. & Pluckthun, A. Engineered proteins as specific binding reagents. Curr. Opin. Biotechnol. 16, 459–469 (2005).

  28. 28

    Skerra, A. Alternative non-antibody scaffolds for molecular recognition. Curr. Opin. Biotechnol. 18, 295–304 (2007).

  29. 29

    Bradbury, A.R. & Marks, J.D. Antibodies from phage antibody libraries. J. Immunol. Methods 290, 29–49 (2004).

  30. 30

    Throsby, M. et al. Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells. PLoS ONE 3, e3942 (2008).

  31. 31

    Razai, A. et al. Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A. J. Mol. Biol. 351, 158–169 (2005).

  32. 32

    Lee, C.V. et al. High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J. Mol. Biol. 340, 1073–1093 (2004).

  33. 33

    Hanes, J., Schaffitzel, C., Knappik, A. & Pluckthun, A. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat. Biotechnol. 18, 1287–1292 (2000).

  34. 34

    Schier, R. et al. 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, 551–567 (1996).

  35. 35

    Yang, W.P. et al. CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J. Mol. Biol. 254, 392–403 (1995).

  36. 36

    Boder, E.T., Midelfort, K.S. & Wittrup, K.D. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. USA 97, 10701–10705 (2000).

  37. 37

    Foote, J. & Eisen, H.N. Breaking the affinity ceiling for antibodies and T cell receptors. Proc. Natl. Acad. Sci. USA 97, 10679–10681 (2000).

  38. 38

    Batista, F.D. & Neuberger, M.S. Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. Immunity 8, 751–759 (1998).

  39. 39

    Schofield, D.J. et al. Application of phage display to high throughput antibody generation and characterization. Genome Biol. 8, R254 (2007).

  40. 40

    Dübel, S., Stoevesandt, O., Taussig, M.J. & Hust, M. Generating recombinant antibodies to the complete human proteome. Trends Biotechnol. 28, 333–339 (2010).

  41. 41

    Koide, A., Bailey, C.W., Huang, X. & Koide, S. The fibronectin type III domain as a scaffold for novel binding proteins. J. Mol. Biol. 284, 1141–1151 (1998).

  42. 42

    Philibert, P. et al. A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm. BMC Biotechnol. 7, 81 (2007).

  43. 43

    Parsons, H.L. et al. Directing phage selections towards specific epitopes. Protein Eng. 9, 1043–1049 (1996).

  44. 44

    Lassen, K.S., Bradbury, A.R., Rehfeld, J.F. & Heegaard, N.H. Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibody. Electrophoresis 29, 2557–2564 (2008).

  45. 45

    Kehoe, J.W. et al. Using phage display to select antibodies recognizing post-translational modifications independently of sequence context. Mol. Cell. Proteomics 5, 2350–2363 (2006).

  46. 46

    Hoffhines, A.J., Damoc, E., Bridges, K.G., Leary, J.A. & Moore, K.L. Detection and purification of tyrosine-sulfated proteins using a novel anti-sulfotyrosine monoclonal antibody. J. Biol. Chem. 281, 37877–37887 (2006).

  47. 47

    Grunewald, J. et al. Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids. Proc. Natl. Acad. Sci. USA 106, 4337–4342 (2009).

  48. 48

    Dalum, I. et al. Therapeutic antibodies elicited by immunization against TNF-alpha. Nat. Biotechnol. 17, 666–669 (1999).

  49. 49

    Hust, M. et al. A human scFv antibody generation pipeline for proteome research. J. Biotechnol. published online, doi:10.1016/j.jbiotec.2010.09.945 (29 September 2010).

  50. 50

    Lloyd, C. et al. 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, 159–168 (2009).

  51. 51

    Wright, K., Collins, D.C. & Preedy, J.R. Comparative specificity of antisera raised against estrone, estradiol-17 and estriol using 6–0-carboxy-methyloxime bovine serum albumin derivatives. Steroids 21, 755–769 (1973).

  52. 52

    Haning, R. et al. The evolution of titer and specificity of aldosterone binding antibodies in hyperimmunized sheep. Steroids 20, 73–88 (1972).

  53. 53

    Exley, D., Johnson, M.W. & Dean, P.D. Antisera highly specific for 17-oestradiol. Steroids 18, 605–620 (1971).

  54. 54

    Tateishi, K., Hamaoka, T., Takatsu, K. & Hayashi, C. A novel immunization procedure for production of anti-testosterone and anti-5 alpha-dihydrotestosterone antisera of low cross-reactivity. J. Steroid Biochem. 13, 951–959 (1980).

  55. 55

    Smith, T.W. & Skubitz, K.M. Kinetics in interactions between antibodies and haptens. Biochemistry 14, 1496–1502 (1975).

  56. 56

    Monigatti, F., Gasteiger, E., Bairoch, A. & Jung, E. The Sulfinator: predicting tyrosine sulfation sites in protein sequences. Bioinformatics 18, 769–770 (2002).

  57. 57

    Sako, D. et al. A sulfated peptide segment at the amino terminus of PSGL-1 is critical for P-selectin binding. Cell 83, 323–331 (1995).

  58. 58

    Rigby, P.W., Gething, M.J. & Hartley, B.S. Construction of intergeneric hybrids using bacteriophage P1CM: transfer of the Klebsiella aerogenes ribitol dehydrogenase gene to Escherichia coli. J. Bacteriol. 125, 728–738 (1976).

  59. 59

    Ayriss, J., Woods, T., Bradbury, A. & Pavlik, P. High-throughput screening of single-chain antibodies using multiplexed flow cytometry. J. Proteome Res. 6, 1072–1082 (2007).

  60. 60

    Pershad, K. et al. Generating a panel of highly specific antibodies to 20 human SH2 domains by phage display. Protein Eng. Des. Sel. 23, 279–288 (2010).

  61. 61

    Mersmann, M. et al. Towards proteome scale antibody selections using phage display. New Biotechnol. 27, 118–128 (2009).

  62. 62

    Velappan, N. et al. Selection and characterization of scFv antibodies against the Sin Nombre hantavirus nucleocapsid protein. J. Immunol. Methods 321, 60–69 (2007).

  63. 63

    Cabezas, S. et al. Phage-displayed antibody fragments recognizing dengue 3 and dengue 4 viruses as tools for viral serotyping in sera from infected individuals. Arch. Virol. 154, 1035–1045 (2009).

  64. 64

    Lim, A.P. et al. Neutralizing human monoclonal antibody against H5N1 influenza HA selected from a Fab-phage display library. Virol. J. 5, 130 (2008).

  65. 65

    Okada, J. et al. 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, 322–330 (2010).

  66. 66

    Meissner, F. et al. Detection of antibodies against the four subtypes of Ebola virus in sera from any species using a novel antibody-phage indicator assay. Virology 300, 236–243 (2002).

  67. 67

    Kirsch, M.I. et al. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV). BMC Biotechnol. 8, 66 (2008).

  68. 68

    Maynard, J.A. et al. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nat. Biotechnol. 20, 597–601 (2002).

  69. 69

    Mabry, R. et al. Passive protection against anthrax by using a high-affinity antitoxin antibody fragment lacking an Fc region. Infect. Immun. 73, 8362–8368 (2005).

  70. 70

    Wild, M.A. et al. Human antibodies from immunized donors are protective against anthrax toxin in vivo. Nat. Biotechnol. 21, 1305–1306 (2003).

  71. 71

    Hayhurst, A. et al. Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis. J. Immunol. Methods 276, 185–196 (2003).

  72. 72

    Zou, N., Newsome, T., Li, B., Tsai, S. & Lo, S.C. Human single-chain Fv antibodies against Burkholderia mallei and Burkholderia pseudomallei. Exp. Biol. Med. 232, 550–556 (2007).

  73. 73

    Steiniger, S.C., Altobell, L.J. III, Zhou, B. & Janda, K.D. Selection of human antibodies against cell surface-associated oligomeric anthrax protective antigen. Mol. Immunol. 44, 2749–2755 (2007).

  74. 74

    Cirino, N.M. et al. Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors. Infect. Immun. 67, 2957–2963 (1999).

  75. 75

    Zhou, B., Wirsching, P. & Janda, K.D. 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, 5241–5246 (2002).

  76. 76

    Garcia-Rodriguez, C. et al. Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin. Nat. Biotechnol. 25, 107–116 (2007).

  77. 77

    Huie, M.A. et al. Antibodies to human fetal erythroid cells from a nonimmune phage antibody library. Proc. Natl. Acad. Sci. USA 98, 2682–2687 (2001).

  78. 78

    Nizak, C. et al. Recombinant antibodies to the small GTPase Rab6 as conformation sensors. Science 300, 984–987 (2003).

  79. 79

    Gao, J., Sidhu, S.S. & Wells, J.A. Two-state selection of conformation-specific antibodies. Proc. Natl. Acad. Sci. USA 106, 3071–3076 (2009).

  80. 80

    Eisenhardt, S.U., Schwarz, M., Bassler, N. & Peter, K. Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules. Nat. Protoc. 2, 3063–3073 (2007).

  81. 81

    Ye, J.D. et al. Synthetic antibodies for specific recognition and crystallization of structured RNA. Proc. Natl. Acad. Sci. USA 105, 82–87 (2008).

  82. 82

    Edwards, B.M. et al. The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS. J. Mol. Biol. 334, 103–118 (2003).

  83. 83

    Baker, K.P. et al. Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator. Arthritis Rheum. 48, 3253–3265 (2003).

  84. 84

    Runnels, H.A. et al. Human monoclonal antibodies to the insulin-like growth factor 1 receptor inhibit receptor activation and tumor growth in preclinical studies. Adv. Ther. 27, 458–475 (2010).

  85. 85

    Peipp, M., Dechant, M. & Valerius, T. Effector mechanisms of therapeutic antibodies against ErbB receptors. Curr. Opin. Immunol. 20, 436–443 (2008).

  86. 86

    Li, K. et al. Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of NOTCH3. J. Biol. Chem. 283, 8046–8054 (2008).

  87. 87

    Wu, Y. et al. Therapeutic antibody targeting of individual Notch receptors. Nature 464, 1052–1057 (2010).

  88. 88

    Martens, T. et al. A novel one-armed anti-c-Met antibody inhibits glioblastoma growth in vivo. Clin. Cancer Res. 12, 6144–6152 (2006).

  89. 89

    Dobson, C.L. et al. Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism. MAbs 1, 552–562 (2009).

  90. 90

    Sui, J. et al. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat. Struct. Mol. Biol. 16, 265–273 (2009).

  91. 91

    Sun, L. et al. Generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza A H5N1 viruses. PLoS ONE 4, e5476 (2009).

  92. 92

    Poul, M.A., Becerril, B., Nielsen, U.B., Morisson, P. & Marks, J.D. Selection of tumor-specific internalizing human antibodies from phage libraries. J. Mol. Biol. 301, 1149–1161 (2000).

  93. 93

    Heitner, T. et al. Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library. J. Immunol. Methods 248, 17–30 (2001).

  94. 94

    Zhou, Y., Zou, H., Zhang, S. & Marks, J.D. Internalizing cancer antibodies from phage libraries selected on tumor cells and yeast-displayed tumor antigens. J. Mol. Biol. 404, 88–99 (2010).

  95. 95

    Park, J.W. et al. Tumor targeting using anti-her2 immunoliposomes. J. Control. Release 74, 95–113 (2001).

  96. 96

    Nielsen, U.B. et al. Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis. Biochim. Biophys. Acta 1591, 109–118 (2002).

  97. 97

    Wu, H. et al. Stepwise in vitro affinity maturation of Vitaxin, an alphav beta3-specific humanized mAb. Proc. Natl. Acad. Sci. USA 95, 6037–6042 (1998).

  98. 98

    Lippow, S.M., Wittrup, K.D. & Tidor, B. Computational design of antibody-affinity improvement beyond in vivo maturation. Nat. Biotechnol. 25, 1171–1176 (2007).

  99. 99

    Fellouse, F.A., Wiesmann, C. & Sidhu, S.S. Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc. Natl. Acad. Sci. USA 101, 12467–12472 (2004).

  100. 100

    Liang, W.C. et al. 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, 951–961 (2006).

  101. 101

    Lee, C.V. et al. Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells. Blood 108, 3103–3111 (2006).

  102. 102

    Fagete, S. et al. Specificity tuning of antibody fragments to neutralize two human chemokines with a single agent. MAbs 1, 288–296 (2009).

  103. 103

    Bostrom, J. et al. Variants of the antibody Herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610–1614 (2009).

  104. 104

    Volk, W.A., Bizzini, B., Snyder, R.M., Bernhard, E. & Wagner, R.R. Neutralization of tetanus toxin by distinct monoclonal antibodies binding to multiple epitopes on the toxin molecule. Infect. Immun. 45, 604–609 (1984).

  105. 105

    Marks, J.D. Deciphering antibody properties that lead to potent botulinum neurotoxin neutralization. Mov. Disord. 19 Suppl 8, S101–S108 (2004).

  106. 106

    Nowakowski, A. et al. Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody. Proc. Natl. Acad. Sci. USA 99, 11346–11350 (2002).

  107. 107

    Kalb, S.R. et al. Extraction of BoNT/A, /B, /E, and /F with a single, high affinity monoclonal antibody for detection of botulinum neurotoxin by Endopep-MS. PLoS ONE 5, e12237 (2010).

  108. 108

    Garcia-Rodriguez, C. et al. Neutralizing human monoclonal antibodies binding multiple serotypes of botulinum neurotoxin. Protein Eng. Des. Sel. published online, doi:10.1093/protein/gzq111 (12 December 2010).

  109. 109

    de Kruif, J. & Logtenberg, T. Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library. J. Biol. Chem. 271, 7630–7634 (1996).

  110. 110

    Hudson, P.J. & Kortt, A.A. High avidity scFv multimers; diabodies and triabodies. J. Immunol. Methods 231, 177–189 (1999).

  111. 111

    Dübel, S. et al. Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv). J. Immunol. Methods 178, 201–209 (1995).

  112. 112

    Griep, R.A. et al. pSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins. Protein Expr. Purif. 16, 63–69 (1999).

  113. 113

    Cloutier, S.M. et al. Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin. Mol. Immunol. 37, 1067–1077 (2000).

  114. 114

    Casey, J.L., Coley, A.M., Tilley, L.M. & Foley, M. Green fluorescent antibodies: novel in vitro tools. Protein Eng. 13, 445–452 (2000).

  115. 115

    Thie, H., Binius, S., Schirrmann, T., Hust, M. & Dübel, S. Multimerization domains for antibody phage display and antibody production. New Biotechnol. 26, 314–321 (2009).

  116. 116

    Persic, L. et al. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187, 9–18 (1997).

  117. 117

    Hu, S. et al. Minibody: A novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts. Cancer Res. 56, 3055–3061 (1996).

  118. 118

    Beck, A. et al. Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. Curr. Pharm. Biotechnol. 9, 482–501 (2008).

  119. 119

    Presta, L.G. Molecular engineering and design of therapeutic antibodies. Curr. Opin. Immunol. 20, 460–470 (2008).

  120. 120

    Merchant, A.M. et al. An efficient route to human bispecific IgG. Nat. Biotechnol. 16, 677–681 (1998).

  121. 121

    Ridgway, J.B., Presta, L.G. & Carter, P. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 9, 617–621 (1996).

  122. 122

    Perisic, O., Webb, P.A., Holliger, P., Winter, G. & Williams, R.L. Crystal structure of a diabody, a bivalent antibody fragment. Structure 2, 1217–1226 (1994).

  123. 123

    Atwell, J.L. et al. 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, 597–604 (1999).

  124. 124

    Pei, X.Y., Holliger, P., Murzin, A.G. & Williams, R.L. 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, 9637–9642 (1997).

  125. 125

    Le Gall, F., Kipriyanov, S.M., Moldenhauer, G. & Little, M. Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Lett. 453, 164–168 (1999).

  126. 126

    Muller, D. & Kontermann, R.E. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 24, 89–98 (2010).

  127. 127

    Xiang, J. Targeting cytokines to tumors to induce active antitumor immune responses by recombinant fusion proteins. Hum. Antibodies 9, 23–36 (1999).

  128. 128

    Schliemann, C. & Neri, D. Antibody-based targeting of the tumor vasculature. Biochim. Biophys. Acta 1776, 175–192 (2007).

  129. 129

    Deckert, P.M. Current constructs and targets in clinical development for antibody-based cancer therapy. Curr. Drug Targets 10, 158–175 (2009).

  130. 130

    Fuchs, H. & Bachran, C. Targeted tumor therapies at a glance. Curr. Drug Targets 10, 89–93 (2009).

  131. 131

    Gawlitta, W., Osborn, M. & Weber, K. Coiling of intermediate filaments induced by microinjection of a vimentin-specific antibody does not interfere with locomotion and mitosis. Eur. J. Cell Biol. 26, 83–90 (1981).

  132. 132

    Kontermann, R.E. Intrabodies as therapeutic agents. Methods 34, 163–170 (2004).

  133. 133

    Beerli, R.R., Wels, W. & Hynes, N.E. Intracellular expression of single chain antibodies reverts ErbB-2 transformation. J. Biol. Chem. 269, 23931–23936 (1994).

  134. 134

    Richardson, J.H., Sodroski, J.G., Waldmann, T.A. & Marasco, W.A. Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor. Proc. Natl. Acad. Sci. USA 92, 3137–3141 (1995).

  135. 135

    Paganetti, P., Calanca, V., Galli, C., Stefani, M. & Molinari, M. beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide. J. Cell Biol. 168, 863–868 (2005).

  136. 136

    Strebe, N. et al. Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies. J. Immunol. Methods 341, 30–40 (2009).

  137. 137

    Biocca, S. & Cattaneo, A. Intracellular immunization: antibody targeting to subcellular compartments. Trends Cell Biol. 5, 248–252 (1995).

  138. 138

    Biocca, S., Pierandrei-Amaldi, P., Campioni, N. & Cattaneo, A. Intracellular immunization with cytosolic recombinant antibodies. Nat. Biotechnol. 12, 396–399 (1994).

  139. 139

    Desiderio, A. et al. A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold. J. Mol. Biol. 310, 603–615 (2001).

  140. 140

    der Maur, A.A. et al. Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework. J. Biol. Chem. 277, 45075–45085 (2002).

  141. 141

    Tanaka, T., Chung, G.T., Forster, A., Lobato, M.N. & Rabbitts, T.H. De novo production of diverse intracellular antibody libraries. Nucleic Acids Res. 31, e23 (2003).

  142. 142

    Auf der Maur, A., Escher, D. & Barberis, A. Antigen-independent selection of stable intracellular single-chain antibodies. FEBS Lett. 508, 407–412 (2001).

  143. 143

    Visintin, M., Tse, E., Axelson, H., Rabbitts, T.H. & Cattaneo, A. Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc. Natl. Acad. Sci. USA 96, 11723–11728 (1999).

  144. 144

    Amstutz, P. et al. Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins. J. Biol. Chem. 280, 24715–24722 (2005).

  145. 145

    Kohl, A. et al. Allosteric inhibition of aminoglycoside phosphotransferase by a designed ankyrin repeat protein. Structure 13, 1131–1141 (2005).

  146. 146

    Rizk, S.S. et al. An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells. Proc. Natl. Acad. Sci. USA 106, 11011–11015 (2009).

  147. 147

    Hallborn, J. & Carlsson, R. Automated screening procedure for high-throughput generation of antibody fragments. Biotechniques Suppl, 30–37 (2002).

  148. 148

    Turunen, L., Takkinen, K., Soderlund, H. & Pulli, T. Automated panning and screening procedure on microplates for antibody generation from phage display libraries. J. Biomol. Screen. 14, 282–293 (2009).

  149. 149

    Lou, J. et al. Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries. J. Immunol. Methods 253, 233–242 (2001).

  150. 150

    Storz, U. IP issues in the therapeutic antibody industry. in Antibody Engineering (eds. Kontermann, R. & Dübel, S.) 517–581 (Springer, 2010).

  151. 151

    Kreitman, R.J. et al. Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies. J. Clin. Oncol. 18, 1622–1636 (2000).

  152. 152

    Fellouse, F.A. et al. High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J. Mol. Biol. 373, 924–940 (2007).

  153. 153

    Hamilton, S.R. & Gerngross, T.U. Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr. Opin. Biotechnol. 18, 387–392 (2007).

  154. 154

    Moghaddam, A. et al. Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine. J. Immunol. Methods 280, 139–155 (2003).

  155. 155

    Dorsam, H. et al. Antibodies to steroids from a small human naive IgM library. FEBS Lett. 414, 7–13 (1997).

  156. 156

    Pope, A. et al. In vitro selection of a high affinity antibody to oestradiol using a phage display human antibody library. Immunotechnology 2, 209–217 (1996).

  157. 157

    Hemminki, A., Niemi, S., Hautoniemi, L., Soderlund, H. & Takkinen, K. Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs. Immunotechnology 4, 59–69 (1998).

  158. 158

    Saviranta, P. et al. Engineering the steroid-specificity of an anti-17beta-estradiol Fab by random mutagenesis and competitive phage panning. Protein Eng. 11, 143–152 (1998).

  159. 159

    Chames, P., Coulon, S. & Baty, D. Improving the affinity and the fine specificity of an anti-cortisol antibody by parsimonious mutagenesis and phage display. J. Immunol. 161, 5421–5429 (1998).

  160. 160

    Bikker, F.J., Mars-Groenendijk, R.H., Noort, D., Fidder, A. & van der Schans, G.P. Detection of sulfur mustard adducts in human callus by phage antibodies. Chem. Biol. Drug Des. 69, 314–320 (2007).

  161. 161

    Szent-Gyorgyi, C. et al. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat. Biotechnol. 26, 235–240 (2008).

  162. 162

    Watanabe, H., Nakanishi, T., Umetsu, M. & Kumagai, I. Human anti-gold antibodies: biofunctionalization of gold nanoparticles and surfaces with anti-gold antibodies. J. Biol. Chem. 283, 36031–36038 (2008).

  163. 163

    Dadaglio, G., Nelson, C.A., Deck, M.B., Petzold, S.J. & Unanue, E.R. Characterization and quantitation of peptide-MHC complexes produced from hen egg lysozyme using a monoclonal antibody. Immunity 6, 727–738 (1997).

  164. 164

    Krogsgaard, M. et al. Visualization of myelin basic protein (MBP) T cell epitopes in multiple sclerosis lesions using a monoclonal antibody specific for the human histocompatibility leukocyte antigen (HLA)-DR2-MBP 85–99 complex. J. Exp. Med. 191, 1395–1412 (2000).

  165. 165

    Mutuberria, R. et al. 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, 31–47 (2004).

  166. 166

    Cohen, C.J., Denkberg, G., Lev, A., Epel, M. & Reiter, Y. 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, 324–332 (2003).

  167. 167

    Engberg, J., Krogsgaard, M. & Fugger, L. Recombinant antibodies with the antigen-specific, MHC restricted specificity of T cells: novel reagents for basic and clinical investigations and immunotherapy. Immunotechnology 4, 273–278 (1999).

  168. 168

    Stryhn, A. et al. Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex. Proc. Natl. Acad. Sci. USA 93, 10338–10342 (1996).

  169. 169

    Villa, A. et al. 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, 2405–2413 (2008).

  170. 170

    Pini, A. et al. 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, 21769–21776 (1998).

  171. 171

    Schliemann, C. & Neri, D. Antibody-based vascular tumor targeting. Recent Results Cancer Res. 180, 201–216 (2010).

  172. 172

    Rothlisberger, D., Pos, K.M. & Pluckthun, A. An antibody library for stabilizing and crystallizing membrane proteins–selecting binders to the citrate carrier CitS. FEBS Lett. 564, 340–348 (2004).

  173. 173

    Uysal, S. et al. Crystal structure of full-length KcsA in its closed conformation. Proc. Natl. Acad. Sci. USA 106, 6644–6649 (2009).

  174. 174

    Osbourn, J., Groves, M. & Vaughan, T. From rodent reagents to human therapeutics using antibody guided selection. Methods 36, 61–68 (2005).

  175. 175

    Jespers, L.S., Roberts, A., Mahler, S.M., Winter, G. & Hoogenboom, H.R. Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. Bio/Technology 12, 899–903 (1994).

  176. 176

    Xie, M.H., Yuan, J., Adams, C. & Gurney, A. Direct demonstration of MuSK involvement in acetylcholine receptor clustering through identification of agonist ScFv. Nat. Biotechnol. 15, 768–771 (1997).

  177. 177

    Ellmark, P., Andersson, H., Abayneh, S., Fenyo, E.M. & Borrebaeck, C.A. Identification of a strongly activating human anti-CD40 antibody that suppresses HIV type 1 infection. AIDS Res. Hum. Retroviruses 24, 367–373 (2008).

  178. 178

    Roth, A. et al. Anti-CD166 single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells. Mol. Cancer Ther. 6, 2737–2746 (2007).

  179. 179

    Liu, B. et al. Recombinant full-length human IgG1s targeting hormone-refractory prostate cancer. J. Mol. Med. 85, 1113–1123 (2007).

Download references

Acknowledgements

A.R.M.B. is grateful to the US National Institutes of Health (P50GM085273 and R01-HG004852-01A1), US Department of Energy (GTL program) and the US Department of Defense, Defense Threat Reduction Agency for funding. S.D. gratefully acknowledges funding by the EU 7th framework programme (Projects: Affinomics and AffinityProteome). J.M. is pleased to acknowledge funding by the Wellcome Trust.

Author information

Correspondence to Andrew R M Bradbury.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Further reading