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  • Review Article
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Engineered antibody fragments and the rise of single domains

Nature Biotechnology volume 23, pages 1126–1136 (2005)Cite this article

Abstract

With 18 monoclonal antibody (mAb) products currently on the market and more than 100 in clinical trials, it is clear that engineered antibodies have come of age as biopharmaceuticals. In fact, by 2008, engineered antibodies are predicted to account for >30% of all revenues in the biotechnology market. Smaller recombinant antibody fragments (for example, classic monovalent antibody fragments (Fab, scFv)) and engineered variants (diabodies, triabodies, minibodies and single-domain antibodies) are now emerging as credible alternatives. These fragments retain the targeting specificity of whole mAbs but can be produced more economically and possess other unique and superior properties for a range of diagnostic and therapeutic applications. Antibody fragments have been forged into multivalent and multispecific reagents, linked to therapeutic payloads (such as radionuclides, toxins, enzymes, liposomes and viruses) and engineered for enhanced therapeutic efficacy. Recently, single antibody domains have been engineered and selected as targeting reagents against hitherto immunosilent cavities in enzymes, receptors and infectious agents. Single-domain antibodies are anticipated to significantly expand the repertoire of antibody-based reagents against the vast range of novel biomarkers being discovered through proteomics. As this review aims to show, there is tremendous potential for all antibody fragments either as robust diagnostic reagents (for example in biosensors), or as nonimmunogenic in vivo biopharmaceuticals with superior biodistribution and blood clearance properties.

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Figure 1: Schematic representation of different antibody formats, showing intact 'classic' IgG molecules alongside camelid VhH-Ig and shark Ig-NAR immunoglobulins.

Katie Ris

Figure 2: Structural comparison of antibody fragments and single domains.
Figure 3: Antibody formats used in imaging.

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References

  1. Better, M., Chang, C.P., Robinson, R.R. & Horwitz, A.H. Escherichia coli secretion of an active chimeric antibody fragment. Science 240, 1041–1043 (1988).

    Article  CAS  PubMed  Google Scholar 

  2. Huston, J.S. et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 5879–5883 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bird, R.E. et al. Single-chain antigen-binding proteins. Science 242, 423–426 (1988).

    Article  CAS  PubMed  Google Scholar 

  4. Holliger, P., Prospero, T. & Winter, G. “Diabodies”: small bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. USA 90, 6444–6448 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Iliades, P., Kortt, A.A. & Hudson, P.J. Triabodies: single chain Fv fragments without a linker form trivalent trimers. FEBS Lett. 409, 437–441 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Ward, E.S., Gussow, D., Griffiths, A.D., Jones, P.T. & Winter, G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544–546 (1989).

    Article  CAS  PubMed  Google Scholar 

  8. Hudson, P.J. & Souriau, C. Engineered antibodies. Nat. Med. 9, 129–134 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Woof, J.M. & Burton, D.R. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat. Rev. Immunol. 4, 89–99 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Ward, E.S. et al. From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases with the Fc receptor, FcRn, during recycling. Mol. Biol. Cell 16, 2028–2038 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cardoso, R.M. et al. Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity 22, 163–173 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Zhang, M.Y. et al. Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody. J. Virol. 78, 9233–9242 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Adams, E.J., Chien, Y.H. & Garcia, K.C. Structure of a gammadelta T cell receptor in complex with the nonclassical MHC T22. Science 308, 227–231 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. De Genst, E. et al. Strong in vivo maturation compensates for structurally restricted H3 loops in antibody repertoires. J. Biol. Chem. 280, 14114–14121 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. De Genst, E. et al. Chemical basis for the affinity maturation of a camel single domain antibody. J. Biol. Chem. 279, 53593–53601 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Dooley, H. & Flajnik, M.F. Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum. Eur. J. Immunol. 35, 936–945 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Streltsov, V. & Nuttall, S. Do sharks have a new antibody lineage? Immunol. Lett. 97, 159–160 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Streltsov, V.A. et al. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor. Proc. Natl. Acad. Sci. USA 101, 12444–12449 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Stanfield, R.L., Dooley, H., Flajnik, M.F. & Wilson, I.A. Crystal structure of a shark single-domain antibody V region in complex with lysozyme. Science 305, 1770–1773 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Nuttall, S.D. et al. Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1. Proteins 55, 187–197 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Conrath, K. et al. Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH. J. Mol. Biol. 350, 112–125 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Saerens, D. et al. Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen. J. Biol. Chem. 279, 51965–51972 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Pleschberger, M. et al. An S-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology. Bioconjug. Chem. 15, 664–671 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Stijlemans, B. et al. Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies. African trypanosomes as paradigm. J. Biol. Chem. 279, 1256–1261 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Cortez-Retamozo, V. et al. Efficient cancer therapy with a nanobody-based conjugate. Cancer Res. 64, 2853–2857 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Dottorini, T., Vaughan, C.K., Walsh, M.A., LoSurdo, P. & Sollazzo, M. Crystal structure of a human VH: requirements for maintaining a monomeric fragment. Biochemistry 43, 622–628 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Colby, D.W. et al. Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display. J. Mol. Biol. 342, 901–912 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Jespers, L., Schon, O., James, L.C., Veprintsev, D. & Winter, G. Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold. J. Mol. Biol. 337, 893–903 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Jespers, L., Schon, O., Famm, K. & Winter, G. Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat. Biotechnol. 22, 1161–1165 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Souriau, C., Chiche, L., Irving, R. & Hudson, P. New binding specificities derived from Min-23, a small cystine-stabilized peptidic scaffold. Biochemistry 44, 7143–7155 (2005).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  32. Midelfort, K.S. et al. Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody. J. Mol. Biol. 343, 685–701 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Moroncini, G. et al. Motif-grafted antibodies containing the replicative interface of cellular PrP are specific for PrPSc. Proc. Natl. Acad. Sci. USA 101, 10404–10409 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. James, L.C., Roversi, P. & Tawfik, D.S. Antibody multispecificity mediated by conformational diversity. Science 299, 1362–1367 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Linsley, P.S. New look at an old costimulator. Nat. Immunol. 6, 231–232 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Teeling, J.L. et al. Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood 104, 1793–1800 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Casey, J.L. et al. Tumour targeting of humanised cross-linked divalent-Fab′ antibody fragments: a clinical phase I/II study. Br. J. Cancer 86, 1401–1410 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Weir, A.N. et al. Formatting antibody fragments to mediate specific therapeutic functions. Biochem. Soc. Trans. 30, 512–516 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Dolezal, O. et al. Single-chain Fv multimers of the anti-neuraminidase antibody NC10: the residue at position 15 in the V(L) domain of the scFv-0 (V(L)-V(H)) molecule is primarily responsible for formation of a tetramer-trimer equilibrium. Protein Eng. 16, 47–56 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Power, B.E., Kortt, A.A. & Hudson, P.J. Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy. Methods Mol. Biol. 207, 335–350 (2003).

    CAS  PubMed  Google Scholar 

  41. Arndt, M.A., Krauss, J. & Rybak, S.M. Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers. FEBS Lett. 578, 257–261 (2004).

    Article  CAS  PubMed  Google Scholar 

  42. Griffiths, G.L. et al. Reagents and methods for PET using bispecific antibody pretargeting and 68Ga-radiolabeled bivalent hapten-peptide-chelate conjugates. J. Nucl. Med. 45, 30–39 (2004).

    CAS  PubMed  Google Scholar 

  43. Olafsen, T. et al. Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications. Protein Eng. Des. Sel. 17, 21–27 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Wittel, U.A. et al. The in vivo characteristics of genetically engineered divalent and tetravalent single-chain antibody constructs. Nucl. Med. Biol. 32, 157–164 (2005).

    Article  CAS  PubMed  Google Scholar 

  45. Le Gall, F., Reusch, U., Little, M. & Kipriyanov, S.M. Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody. Protein Eng. Des. Sel. 17, 357–366 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Rosenthal, P.B., Waddington, L.J. & Hudson, P.J. Structure of an influenza neuraminidase-diabody complex by electron cryomicroscopy and image analysis. J. Mol. Biol. 334, 721–731 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Carmichael, J.A. et al. The crystal structure of an anti-CEA scFv diabody assembled from T84.66 scFvs in V(L)-to-V(H) orientation: implications for diabody flexibility. J. Mol. Biol. 326, 341–351 (2003).

    Article  CAS  PubMed  Google Scholar 

  48. Kenanova, V. et al. Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments. Cancer Res. 65, 622–631 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Robinson, M.K. et al. Quantitative immuno-positron emission tomography imaging of HER2-positive tumor xenografts with an iodine-124 labeled anti-HER2 diabody. Cancer Res. 65, 1471–1478 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. Sundaresan, G. et al. 124I-labeled engineered anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice. J. Nucl. Med. 44, 1962–1969 (2003).

    CAS  PubMed  Google Scholar 

  51. Jain, R.K. & Baxter, L.T. Mechanisms of heterogeneous distribution of monoclonal antibodies and other macromolecules in tumors: significance of elevated interstitial pressure. Cancer Res. 48, 7022–7032 (1988).

    CAS  PubMed  Google Scholar 

  52. Rao, B.M., Lauffenburger, D.A. & Wittrup, K.D. Integrating cell-level kinetic modeling into the design of engineered protein therapeutics. Nat. Biotechnol. 23, 191–194 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Oh, P. et al. Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Nature 429, 629–635 (2004).

    Article  CAS  PubMed  Google Scholar 

  54. Adams, G.P. et al. A single treatment of yttrium-90-labeled CHX-A”-C6.5 diabody inhibits the growth of established human tumor xenografts in immunodeficient mice. Cancer Res. 64, 6200–6206 (2004).

    Article  CAS  PubMed  Google Scholar 

  55. Matter, C.M. et al. Molecular imaging of atherosclerotic plaques using a human antibody against the extra-domain B of fibronectin. Circ. Res. 95, 1225–1233 (2004).

    Article  CAS  PubMed  Google Scholar 

  56. Brack, S.S., Dinkelborg, L.M. & Neri, D. Molecular targeting of angiogenesis for imaging and therapy. Eur. J. Nucl. Med. Mol. Imaging 31, 1327–1341 (2004).

    Article  PubMed  Google Scholar 

  57. Olafsen, T. et al. Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting. Protein Eng. Des. Sel. 17, 315–323 (2004).

    Article  CAS  PubMed  Google Scholar 

  58. Nielsen, U.B., Adams, G.P., Weiner, L.M. & Marks, J.D. Targeting of bivalent anti-ErbB2 diabody antibody fragments to tumor cells is independent of the intrinsic antibody affinity. Cancer Res. 60, 6434–6440 (2000).

    CAS  PubMed  Google Scholar 

  59. Fujimori, K., Fisher, D.R. & Weinstein, J.N. Integrated microscopic-macroscopic pharmacology of monoclonal antibody radioconjugates: the radiation dose distribution. Cancer Res. 51, 4821–4827 (1991).

    CAS  PubMed  Google Scholar 

  60. Knight, D.M. et al. Pharmacodynamic enhancement of the anti-platelet antibody fab abciximab by site-specific pegylation. Platelets 15, 409–418 (2004).

    Article  CAS  PubMed  Google Scholar 

  61. Huhalov, A. & Chester, K.A. Engineered single chain antibody fragments for radioimmunotherapy. Q. J. Nucl. Med. Mol. Imaging 48, 279–288 (2004).

    CAS  PubMed  Google Scholar 

  62. Holliger, P., Wing, M., Pound, J.D., Bohlen, H. & Winter, G. Retargeting serum immunoglobulin with bispecific diabodies. Nat. Biotechnol. 15, 632–636 (1997).

    Article  CAS  PubMed  Google Scholar 

  63. Medzihradszky, K.F. et al. Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein. Glycobiology 14, 27–37 (2004).

    Article  CAS  PubMed  Google Scholar 

  64. Sanz, L., Blanco, B. & Alvarez-Vallina, L. Antibodies and gene therapy: teaching old 'magic bullets' new tricks. Trends Immunol. 25, 85–91 (2004).

    Article  CAS  PubMed  Google Scholar 

  65. Afanasieva, T.A. et al. Single-chain antibody and its derivatives directed against vascular endothelial growth factor: application for antiangiogenic gene therapy. Gene Ther. 10, 1850–1859 (2003).

    Article  CAS  PubMed  Google Scholar 

  66. Blanco, B., Holliger, P., Vile, R.G. & Alvarez-Vallina, L. Induction of human T lymphocyte cytotoxicity and inhibition of tumor growth by tumor-specific diabody-based molecules secreted from gene-modified bystander cells. J. Immunol. 171, 1070–1077 (2003).

    Article  CAS  PubMed  Google Scholar 

  67. Lobato, M.N. & Rabbitts, T.H. Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules. Curr. Mol. Med. 4, 519–528 (2004).

    Article  CAS  PubMed  Google Scholar 

  68. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  69. Colby, D.W. et al. Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. Proc. Natl. Acad. Sci. USA 101, 17616–17621 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Jendreyko, N. et al. Intradiabodies, bispecific, tetravalent antibodies for the simultaneous functional knockout of two cell surface receptors. J. Biol. Chem. 278, 47812–47819 (2003).

    Article  CAS  PubMed  Google Scholar 

  71. Zhao, Y., Brown, T.L., Kohler, H. & Muller, S. MTS-conjugated-antiactive caspase 3 antibodies inhibit actinomycin D-induced apoptosis. Apoptosis 8, 631–637 (2003).

    Article  CAS  PubMed  Google Scholar 

  72. Weisbart, R.H. et al. Nuclear delivery of p53 C-terminal peptides into cancer cells using scFv fragments of a monoclonal antibody that penetrates living cells. Cancer Lett. 195, 211–219 (2003).

    Article  CAS  PubMed  Google Scholar 

  73. Carter, P. Bispecific human IgG by design. J. Immunol. Methods 248, 7–15 (2001).

    Article  CAS  PubMed  Google Scholar 

  74. Xie, Z., Guo, N., Yu, M., Hu, M. & Shen, B. A new format of bispecific antibody: highly efficient heterodimerization, expression and tumor cell lysis. J. Immunol. Methods 296, 95–101 (2005).

    Article  CAS  PubMed  Google Scholar 

  75. Zhu, Z., Presta, L.G., Zapata, G. & Carter, P. Remodeling domain interfaces to enhance heterodimer formation. Protein Sci. 6, 781–788 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Schlereth, B. et al. Eradication of tumors from a human colon cancer cell line and from ovarian cancer metastases in immunodeficient mice by a single-chain Ep-CAM-/CD3-bispecific antibody construct. Cancer Res. 65, 2882–2889 (2005).

    Article  CAS  PubMed  Google Scholar 

  77. Hoffmann, P. et al. Serial killing of tumor cells by cytotoxic T cells redirected with a CD19-/CD3-bispecific single-chain antibody construct. Int. J. Cancer 115, 98–104 (2005).

    Article  CAS  PubMed  Google Scholar 

  78. Kipriyanov, S.M. & Le Gall, F. Recent advances in the generation of bispecific antibodies for tumor immunotherapy. Curr. Opin. Drug Discov. Devel. 7, 233–242 (2004).

    CAS  PubMed  Google Scholar 

  79. Schlenzka, J. et al. Combined effect of recombinant CD19 × CD16 diabody and thalidomide in a preclinical model of human B cell lymphoma. Anticancer Drugs 15, 915–919 (2004).

    Article  CAS  PubMed  Google Scholar 

  80. Haas, C., Lulei, M., Fournier, P., Arnold, A. & Schirrmacher, V. T-cell triggering by CD3- and CD28-binding molecules linked to a human virus-modified tumor cell vaccine. Vaccine 23, 2439–2453 (2005).

    Article  CAS  PubMed  Google Scholar 

  81. Lu, D. et al. A fully human recombinant IgG-like bispecific antibody to both the epidermal growth factor receptor and the insulin-like growth factor receptor for enhanced antitumor activity. J. Biol. Chem. 280, 19665–19672 (2005).

    Article  CAS  PubMed  Google Scholar 

  82. Jimenez, X. et al. A recombinant, fully human, bispecific antibody neutralizes the biological activities mediated by both vascular endothelial growth factor receptors 2 and 3. Mol. Cancer Ther. 4, 427–434 (2005).

    Article  CAS  PubMed  Google Scholar 

  83. Korn, T., Nettelbeck, D.M., Volkel, T., Muller, R. & Kontermann, R.E. Recombinant bispecific antibodies for the targeting of adenoviruses to CEA-expressing tumour cells: a comparative analysis of bacterially expressed single-chain diabody and tandem scFv. J. Gene Med. 6, 642–651 (2004).

    Article  CAS  PubMed  Google Scholar 

  84. Schnyder, A. & Huwyler, J. Drug transport to brain with targeted liposomes. NeuroRx 2, 99–107 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  85. Biburger, M., Weth, R. & Wels, W.S. A novel bispecific tetravalent antibody fusion protein to target costimulatory activity for T-cell activation to tumor cells overexpressing ErbB2/HER2. J. Mol. Biol. 346, 1299–1311 (2005).

    Article  CAS  PubMed  Google Scholar 

  86. Sharkey, R.M. & Goldenberg, D.M. Perspectives on cancer therapy with radiolabeled monoclonal antibodies. J. Nucl. Med. 46 (Suppl. 1), 115S–127S (2005).

    CAS  PubMed  Google Scholar 

  87. Zhang, M. et al. Pretargeting radioimmunotherapy of a murine model of adult T-cell leukemia with the alpha-emitting radionuclide, bismuth 213. Blood 100, 208–216 (2002).

    Article  CAS  PubMed  Google Scholar 

  88. Nakamura, T. et al. Antibody-targeted cell fusion. Nat. Biotechnol. 22, 331–336 (2004).

    Article  CAS  PubMed  Google Scholar 

  89. Nakamura, T. et al. Rescue and propagation of fully retargeted oncolytic measles viruses. Nat. Biotechnol. 23, 209–214 (2005).

    Article  CAS  PubMed  Google Scholar 

  90. Bang, S., Nagata, S., Onda, M., Kreitman, R.J. & Pastan, I. HA22 (R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity. Clin. Cancer Res. 11, 1545–1550 (2005).

    Article  CAS  PubMed  Google Scholar 

  91. Vallera, D.A. et al. Molecular modification of a recombinant, bivalent anti-human CD3 immunotoxin (Bic3) results in reduced in vivo toxicity in mice. Leuk. Res. 29, 331–341 (2005).

    Article  CAS  PubMed  Google Scholar 

  92. Krauss, J., Arndt, M.A., Vu, B.K., Newton, D.L. & Rybak, S.M. Targeting malignant B-cell lymphoma with a humanized anti-CD22 scFv-angiogenin immunoenzymedouble dagger. Br. J. Haematol. 128, 602–609 (2005).

    Article  CAS  PubMed  Google Scholar 

  93. Halin, C. et al. Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature. Nat. Biotechnol. 20, 264–269 (2002).

    Article  CAS  PubMed  Google Scholar 

  94. Smallshaw, J.E. et al. Genetic engineering of an immunotoxin to eliminate pulmonary vascular leak in mice. Nat. Biotechnol. 21, 387–391 (2003).

    Article  CAS  PubMed  Google Scholar 

  95. Forsberg, G. et al. Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen. Br. J. Cancer 85, 129–136 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Enever, C., Tomlinson, I.M., Lund, J., Levens, M. & Holliger, P. Engineering high affinity superantigens by phage display. J. Mol. Biol. 347, 107–120 (2005).

    Article  CAS  PubMed  Google Scholar 

  97. Brignole, C. et al. Immune cell-mediated antitumor activities of GD2-targeted liposomal c-myb antisense oligonucleotides containing CpG motifs. J. Natl. Cancer Inst. 96, 1171–1180 (2004).

    Article  CAS  PubMed  Google Scholar 

  98. van Broekhoven, C.L., Parish, C.R., Demangel, C., Britton, W.J. & Altin, J.G. Targeting dendritic cells with antigen-containing liposomes: a highly effective procedure for induction of antitumor immunity and for tumor immunotherapy. Cancer Res. 64, 4357–4365 (2004).

    Article  CAS  PubMed  Google Scholar 

  99. Lu, M., Isogawa, M., Xu, Y. & Hilken, G. Immunization with the gene expressing woodchuck hepatitis virus nucleocapsid protein fused to cytotoxic-T-lymphocyte-associated antigen 4 leads to enhanced specific immune responses in mice and woodchucks. J. Virol. 79, 6368–6376 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Sharma, S.K. et al. Sustained tumor regression of human colorectal cancer xenografts using a multifunctional mannosylated fusion protein in antibody-directed enzyme prodrug therapy. Clin. Cancer Res. 11, 814–825 (2005).

    CAS  PubMed  Google Scholar 

  101. Mayer, A. et al. Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT). Br. J. Cancer 90, 2402–2410 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Mosquera, L.A. et al. In vitro and in vivo characterization of a novel antibody-like single-chain TCR human IgG1 fusion protein. J. Immunol. 174, 4381–4388 (2005).

    Article  CAS  PubMed  Google Scholar 

  103. Michalet, X. et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307, 538–544 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Evans, E.J. et al. Crystal structure of a soluble CD28-Fab complex. Nat. Immunol. 6, 271–279 (2005).

    Article  CAS  PubMed  Google Scholar 

  105. Ravn, P. et al. Multivalent scFv display of phagemid repertoires for the selection of carbohydrate-specific antibodies and its application to the Thomsen-Friedenreich antigen. J. Mol. Biol. 343, 985–996 (2004).

    Article  CAS  PubMed  Google Scholar 

  106. Jespers, L., Bonnert, T.P. & Winter, G. Selection of optical biosensors from chemisynthetic antibody libraries. Protein Eng. Des. Sel. 17, 709–713 (2004).

    Article  CAS  PubMed  Google Scholar 

  107. Sepp, A., Tawfik, D.S. & Griffiths, A.D. Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry. FEBS Lett. 532, 455–458 (2002).

    Article  CAS  PubMed  Google Scholar 

  108. de Wildt, R.M., Tomlinson, I.M., Ong, J.L. & Holliger, P. Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes. Proc. Natl. Acad. Sci. USA 99, 8530–8535 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Dolk, E. et al. Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen. Proteins 59, 555–564 (2005).

    Article  CAS  PubMed  Google Scholar 

  110. Dolk, E. et al. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo. Appl. Environ. Microbiol. 71, 442–450 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Nuttall, S.D. et al. Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70. Eur. J. Biochem. 270, 3543–3554 (2003).

    Article  CAS  PubMed  Google Scholar 

  112. Tanaka, T., Lobato, M.N. & Rabbitts, T.H. Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies. J. Mol. Biol. 331, 1109–1120 (2003).

    Article  CAS  PubMed  Google Scholar 

  113. Wunderlich, M., Martin, A. & Schmid, F.X. Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions. J. Mol. Biol. 347, 1063–1076 (2005).

    Article  CAS  PubMed  Google Scholar 

  114. Dill, K. et al. Immunoassays based on electrochemical detection using microelectrode arrays. Biosens. Bioelectron. 20, 736–742 (2004).

    Article  CAS  PubMed  Google Scholar 

  115. Hamelinck, D. et al. Optimized normalization for antibody microarrays and application to serum-protein profiling. Mol. Cell Proteomics 4, 773–784 (2005).

    Article  CAS  PubMed  Google Scholar 

  116. Orntoft, T.F. & Vestergaard, E.M. Clinical aspects of altered glycosylation of glycoproteins in cancer. Electrophoresis 20, 362–371 (1999).

    Article  CAS  PubMed  Google Scholar 

  117. Prescher, J.A., Dube, D.H. & Bertozzi, C.R. Chemical remodelling of cell surfaces in living animals. Nature 430, 873–877 (2004).

    Article  CAS  PubMed  Google Scholar 

  118. Chambers, R.S. High-throughput antibody production. Curr. Opin. Chem. Biol. 9, 46–50 (2005).

    Article  CAS  PubMed  Google Scholar 

  119. Grosse-Hovest, L. et al. Supraagonistic, bispecific single-chain antibody purified from the serum of cloned, transgenic cows induces T-cell-mediated killing of glioblastoma cells in vitro and in vivo. Int. J. Cancer; published online 7 July 2005. http://www3.interscience.wiley.com/cgi-bin/abstract/110559371/ABSTRACT

  120. Holliger, P. et al. Carcinoembryonic antigen (CEA)-specific T-cell activation in colon carcinoma induced by anti-CD3xanti-CEA bispecific diabodies and B7xantiCEA bspecific fusion proteins. Cancer Res. 59, 2909–2916 (1999).

    CAS  PubMed  Google Scholar 

  121. Pattersen, E.F. et al. UCFS Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    Article  CAS  Google Scholar 

  122. Olafsen, T. et al. Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging. Cancer Res. 65, 5907–5916 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Shen, S. et al. Patient-specific dosimetry of pretargeted radioimmunotherapy using CC49 fusion protein in patients with gastrointestinal malignancies. J. Nucl. Med. 46, 642–651 (2005).

    CAS  PubMed  Google Scholar 

  124. Nellis, D.F. et al. Preclinical manufacture of anti-HER2 liposome-inserting, scFv-PEG-lipid conjugate. 2. Conjugate micelle identity, purity, stability, and potency analysis. Biotechnol. Prog. 21, 221–232 (2005).

    Article  CAS  PubMed  Google Scholar 

  125. Ebbinghaus, C. et al. Engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy. Int. J. Cancer 116, 304–313 (2005).

    Article  CAS  PubMed  Google Scholar 

  126. Wong, J.Y. et al. Pilot trial evaluating an 123I-labeled 80-kilodalton engineered anticarcinoembryonic antigen antibody fragment (cT84.66 minibody) in patients with colorectal cancer. Clin. Cancer Res. 10, 5014–5021 (2004).

    Article  CAS  PubMed  Google Scholar 

  127. Hulstein, J.J. et al. A novel nanobody that detects the gain-of-function phenotype of Von Willebrand factor in adamts13 deficiency and Von Willebrand disease type 2B. Blood 12 July 2005. http://www.bloodjournal.org/cgi/reprint/2005-03-1153v1

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Acknowledgements

We thank V. Streltsov, S. Nuttall and J. Atwell for their help in assembling Figures 1 and 2 and A. Wu and G. Adams for allowing us to include their unpublished data in Figure 3. We also thank our colleagues L. Jespers and I. Tomlinson from Domantis Ltd. for sharing unpublished results and L. James (Medical Research Council) for helpful discussion.

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  1. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Philipp Holliger

  2. CSIRO Molecular and Health Technologies & Cooperative Research Centre for Diagnostics, 343 Royal Parade, Parkville, 3052, Victoria, Australia

    Peter J Hudson

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Correspondence to Philipp Holliger or Peter J Hudson.

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Holliger, P., Hudson, P. Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23, 1126–1136 (2005). https://doi.org/10.1038/nbt1142

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