Advertisement

Alternative Scaffolds as Bispecific Antibody Mimetics

  • John Löfblom
  • Fredrik Y. Frejd
Chapter

Abstract

The use of non-immunoglobulin-based protein scaffolds for engineering of specific recognition was first described some 15 years ago and has matured as a discipline in parallel with the rapidly expanding monoclonal antibody field. As bispecific antibodies and antibody fragments have come into focus lately, the corresponding development of bispecific alternative scaffolds is also emerging. Here, the concept of alternative scaffold proteins is introduced along with comparisons to the antibody and antibody derivatives counterparts. Although the field of bispecifics is anticipated to grow rapidly in the near future, relatively few examples of bispecific binders based on alternative protein scaffolds are reported in the literature. This chapter will present an overview of work that have been published, including a brief introduction to the particular molecular scaffolds with the structural basis and some of the biophysical and biochemical properties before describing the bispecific application.

Keywords

Epidermal Growth Factor Receptor Phage Display Diabetic Macular Edema Bispecific Antibody Affibody Molecule 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Ahlgren S, Orlova A, Wållberg H, Hansson M, Sandström M, Lewsley R, Wennborg A, Abrahmsén L, Tolmachev V, Feldwisch J (2010) Targeting of HER2-expressing tumors using 111In-ABY-025, a second-generation affibody molecule with a fundamentally reengineered scaffold. J Nucl Med 51:1131–1138PubMedCrossRefGoogle Scholar
  2. Alm T, Yderland L, Nilvebrant J, Halldin A, Hober S (2010) A small bispecific protein selected for orthogonal affinity purification. Biotechnol J 5:605–617PubMedCrossRefGoogle Scholar
  3. Bertschinger J, Grabulovski D, Neri D (2007) Selection of single domain binding proteins by covalent DNA display. Protein Eng Des Sel 20:57–68PubMedCrossRefGoogle Scholar
  4. Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, Grutter MG, Plückthun A (2004) High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol 22:575–582PubMedCrossRefGoogle Scholar
  5. Deisenhofer J (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry 20:2361–2370PubMedCrossRefGoogle Scholar
  6. Dincbas-Renqvist V, Lendel C, Dogan J, Wahlberg E, Härd T (2004) Thermodynamics of folding, stabilization, and binding in an engineered protein–protein complex. J Am Chem Soc 126:11220–11230PubMedCrossRefGoogle Scholar
  7. Dogan J, Lendel C, Hard T (2006) Thermodynamics of folding and binding in an affibody:affibody complex. J Mol Biol 359:1305–1315PubMedCrossRefGoogle Scholar
  8. Ebersbach H, Fiedler E, Scheuermann T, Fiedler M, Stubbs MT, Reimann C, Proetzel G, Rudolph R, Fiedler U (2007) Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein. J Mol Biol 372:172–185PubMedCrossRefGoogle Scholar
  9. Eggel A, Baumann MJ, Amstutz P, Stadler BM, Vogel M (2009) DARPins as bispecific receptor antagonists analyzed for immunoglobulin E receptor blockage. J Mol Biol 393:598–607PubMedCrossRefGoogle Scholar
  10. Eigenbrot C, Ultsch M, Dubnovitsky A, Abrahmsén L, Härd T (2010) Structural basis for high-affinity HER2 receptor binding by an engineered protein. Proc Natl Acad Sci USA 107:15039–15044PubMedCrossRefGoogle Scholar
  11. Emanuel SL et al. (2011) A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor. MAbs 3:38–48PubMedCrossRefGoogle Scholar
  12. Engfeldt T, Renberg B, Brumer H, Nygren PÅ, Karlström AE (2005) Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein. Chembiochem 6:1043–1050PubMedCrossRefGoogle Scholar
  13. Engfeldt T, Orlova A, Tran T, Bruskin A, Widstrom C, Karlström AE, Tolmachev V (2007) Imaging of HER2-expressing tumours using a synthetic affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence. Eur J Nucl Med Mol Imaging 34:722–733PubMedCrossRefGoogle Scholar
  14. Feldwisch J, Tolmachev V, Lendel C, Herne N, Sjöberg A, Larsson B, Rosik D, Lindqvist E, Fant G, Höidén-Guthenberg I et al (2010) Design of an optimized scaffold for affibody molecules. J Mol Biol 398:232–247PubMedCrossRefGoogle Scholar
  15. Friedman M, Nordberg E, Höidén-Guthenberg I, Brismar H, Adams GP, Nilsson FY, Carlsson J, Ståhl S (2007) Phage display selection of affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor. Protein Eng Des Sel 20:189–199PubMedCrossRefGoogle Scholar
  16. Friedman M, Orlova A, Johansson E, Eriksson TL, Höidén-Guthenberg I, Tolmachev V, Nilsson FY, Ståhl S (2008) Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule. J Mol Biol 376:1388–1402PubMedCrossRefGoogle Scholar
  17. Friedman M, Lindström S, Ekerljung L, Andersson-Svahn H, Carlsson J, Brismar H, Gedda L, Frejd FY, Ståhl S (2009) Engineering and characterization of a bispecific HER2 x EGFR-binding affibody molecule. Biotechnol Appl Biochem 54:121–131PubMedCrossRefGoogle Scholar
  18. Gebauer M, Skerra A (2009) Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245–255PubMedCrossRefGoogle Scholar
  19. Grönwall C, Jonsson A, Lindström S, Gunneriusson E, Ståhl S, Herne N (2007) Selection and characterization of affibody ligands binding to Alzheimer amyloid beta peptides. J Biotechnol 128:162–183PubMedCrossRefGoogle Scholar
  20. Hogbom M, Eklund M, Nygren PÅ, Nordlund P (2003) Structural basis for recognition by an in vitro evolved affibody. Proc Natl Acad Sci USA 100:3191–3196PubMedCrossRefGoogle Scholar
  21. Hopp J, Hornig N, Zettlitz KA, Schwarz A, Fuss N, Muller D, Kontermann RE (2010) The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein. Protein Eng Des Sel. 23:827–834PubMedGoogle Scholar
  22. Hufton SE, van Neer N, van den Beuken T, Desmet J, Sablon E, Hoogenboom HR (2000) Development and application of cytotoxic T lymphocyte-associated antigen 4 as a protein scaffold for the generation of novel binding ligands. FEBS Lett 475:225–231PubMedCrossRefGoogle Scholar
  23. James LC, Roversi P, Tawfik DS (2003) Antibody multispecificity mediated by conformational diversity. Science 299:1362–1367PubMedCrossRefGoogle Scholar
  24. Jonsson A, Dogan J, Herne N, Abrahmsén L, Nygren PÅ (2008) Engineering of a femtomolar affinity binding protein to human serum albumin. Protein Eng Des Sel 21:515–527PubMedCrossRefGoogle Scholar
  25. Kim HJ, Eichinger A, Skerra A (2009) High-affinity recognition of lanthanide(III) chelate complexes by a reprogrammed human lipocalin 2. J Am Chem Soc 131:3565–3576PubMedCrossRefGoogle Scholar
  26. Koide A, Bailey CW, Huang X, Koide S (1998) The fibronectin type III domain as a scaffold for novel binding proteins. J Mol Biol 284:1141–1151PubMedCrossRefGoogle Scholar
  27. Koide A, Gilbreth RN, Esaki K, Tereshko V, Koide S (2007) High-affinity single-domain binding proteins with a binary-code interface. Proc Natl Acad Sci USA 104:6632–6637PubMedCrossRefGoogle Scholar
  28. Kolmar H (2009) Biological diversity and therapeutic potential of natural and engineered cystine knot miniproteins. Curr Opin Pharmacol 9:608–614PubMedCrossRefGoogle Scholar
  29. Kong A, Calleja V, Leboucher P, Harris A, Parker PJ, Larijani B (2008) HER2 oncogenic function escapes EGFR tyrosine kinase inhibitors via activation of alternative HER receptors in breast cancer cells. PLoS One 3. doi: 10.1371/journal.pone.0002881 Google Scholar
  30. Lehmann A (2008) Ecallantide (DX-88), a plasma kallikrein inhibitor for the treatment of hereditary angioedema and the prevention of blood loss in on-pump cardiothoracic surgery. Expert Opin Biol Ther 8:1187–1199PubMedCrossRefGoogle Scholar
  31. Lendel C, Dincbas-Renqvist V, Flores A, Wahlberg E, Dogan J, Nygren PA, Härd T (2004) Biophysical characterization of Z(SPA-1)–a phage-display selected binder to protein A. Protein Sci 13:2078–2088PubMedCrossRefGoogle Scholar
  32. Lendel C, Dogan J, Härd T (2006) Structural basis for molecular recognition in an affibody:affibody complex. J Mol Biol 359:1293–1304PubMedCrossRefGoogle Scholar
  33. Lipovsek D, Lippow SM, Hackel BJ, Gregson MW, Cheng P, Kapila A, Wittrup KD (2007) Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display: molecular convergence with single-domain camelid and shark antibodies. J Mol Biol 368:1024–1041PubMedCrossRefGoogle Scholar
  34. Löfblom J, Feldwisch J, Tolmachev V, Carlsson J, Ståhl S, Frejd FY (2010) Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett 584:2670–2680PubMedCrossRefGoogle Scholar
  35. Milstein C, Cuello AC (1983) Hybrid hybridomas and their use in immunohistochemistry. Nature 305:537–540PubMedCrossRefGoogle Scholar
  36. Mouquet H, Scheid JF, Zoller MJ, Krogsgaard M, Ott RG, Shukair S, Artyomov MN, Pietzsch J, Connors M, Pereyra F et al (2010) Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation. Nature 467:591–595PubMedCrossRefGoogle Scholar
  37. Nilsson FY, Tolmachev V (2007) Affibody molecules: new protein domains for molecular imaging and targeted tumor therapy. Curr Opin Drug Discov Devel 10:167–175PubMedGoogle Scholar
  38. Nord K, Nilsson J, Nilsson B, Uhlén M, Nygren PÅ (1995) A combinatorial library of an alpha-helical bacterial receptor domain. Protein Eng 8:601–608PubMedCrossRefGoogle Scholar
  39. Nord K, Gunneriusson E, Ringdahl J, Ståhl S, Uhlén M, Nygren PÅ (1997) Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nat Biotechnol 15:772–777PubMedCrossRefGoogle Scholar
  40. Nord K, Nord O, Uhlén M, Kelley B, Ljungqvist C, Nygren PÅ (2001) Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A. Eur J Biochem 268:4269–4277PubMedCrossRefGoogle Scholar
  41. Nygren PÅ, Skerra A (2004) Binding proteins from alternative scaffolds. J Immunol Methods 290:3–28PubMedCrossRefGoogle Scholar
  42. Orlova A, Magnusson M, Eriksson TL, Nilsson M, Larsson B, Höidén-Guthenberg I, Widström C, Carlsson J, Tolmachev V, Ståhl S et al (2006) Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer Res 66:4339–4348PubMedCrossRefGoogle Scholar
  43. Orlova A, Tran T, Widström C, Engfeldt T, Eriksson Karlström A, Tolmachev V (2007a) Pre-clinical evaluation of [111In]-benzyl-DOTA-Z(HER2:342), a potential agent for imaging of HER2 expression in malignant tumors. Int J Mol Med 20:397–404PubMedGoogle Scholar
  44. Orlova A, Tolmachev V, Pehrson R, Lindborg M, Tran T, Sandstrom M, Nilsson FY, Wennborg A, Abrahmsen L, Feldwisch J (2007b) Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors. Cancer Res 67:2178–2186PubMedCrossRefGoogle Scholar
  45. Osaki A, Toi M, Yamada H, Kawami H, Kuroi K, Toge T (1992) Prognostic significance of co-expression of c-erbB-2 oncoprotein and epidermal growth factor receptor in breast cancer patients. Am J Surg 164:323–326PubMedCrossRefGoogle Scholar
  46. Sandström K, Xu Z, Forsberg G, Nygren PÅ (2003) Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding affibody ligand developed by combinatorial protein engineering. Protein Eng 16:691–697PubMedCrossRefGoogle Scholar
  47. Schlehuber S, Skerra A (2001) Duocalins: engineered ligand-binding proteins with dual specificity derived from the lipocalin fold. Biol Chem 382:1335–1342PubMedCrossRefGoogle Scholar
  48. Schlehuber S, Skerra A (2005) Lipocalins in drug discovery: from natural ligand-binding proteins to “anticalins”. Drug Discov Today 10:23–33PubMedCrossRefGoogle Scholar
  49. Silverman J, Liu Q, Bakker A, To W, Duguay A, Alba BM, Smith R, Rivas A, Li P, Le H et al (2005) Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol 23:1556–1561PubMedCrossRefGoogle Scholar
  50. Skerra A (2008) Alternative binding proteins: anticalins – harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities. FEBS J 275:2677–2683PubMedCrossRefGoogle Scholar
  51. Steffen AC, Orlova A, Wikman M, Nilsson FY, Ståhl S, Adams GP, Tolmachev V, Carlsson J (2006) Affibody-mediated tumour targeting of HER-2 expressing xenografts in mice. Eur J Nucl Med Mol Imaging 33:631–638PubMedCrossRefGoogle Scholar
  52. Steiner D, Forrer P, Stumpp MT, Plückthun A (2006) Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nat Biotechnol 24:823–831PubMedCrossRefGoogle Scholar
  53. Stork R, Campigna E, Robert B, Muller D, Kontermann RE (2009) Biodistribution of a bispecific single-chain diabody and its half-life extended derivatives. J Biol Chem 284:25612–25619PubMedCrossRefGoogle Scholar
  54. Stumpp MT, Binz HK, Amstutz P (2008) DARPins: a new generation of protein therapeutics. Drug Discov Today 13:695–701PubMedCrossRefGoogle Scholar
  55. Tolmachev V, Nilsson FY, Widström C, Andersson K, Rosik D, Gedda L, Wennborg A, Orlova A (2006) 111In-benzyl-DTPA-ZHER2:342, an affibody-based conjugate for in vivo imaging of HER2 expression in malignant tumors. J Nucl Med 47:846–853PubMedGoogle Scholar
  56. Tolmachev V, Orlova A, Nilsson FY, Feldwisch J, Wennborg A, Abrahmsén L (2007a) Affibody molecules: potential for in vivo imaging of molecular targets for cancer therapy. Expert Opin Biol Ther 7:555–568PubMedCrossRefGoogle Scholar
  57. Tolmachev V, Orlova A, Pehrson R, Galli J, Baastrup B, Andersson K, Sandström M, Rosik D, Carlsson J, Lundqvist H et al (2007b) Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific affibody molecule. Cancer Res 67:2773–2782PubMedCrossRefGoogle Scholar
  58. Tsutsui S, Ohno S, Murakami S, Kataoka A, Kinoshita J, Hachitanda Y (2003) Prognostic value of the combination of epidermal growth factor receptor and c-erbB-2 in breast cancer. Surgery 133:219–221PubMedCrossRefGoogle Scholar
  59. Wahlberg E, Lendel C, Helgstrand M, Allard P, Dincbas-Renqvist V, Hedqvist A, Berglund H, Nygren PÅ, Härd T (2003) An affibody in complex with a target protein: structure and coupled folding. Proc Natl Acad Sci USA 100:3185–3190PubMedCrossRefGoogle Scholar
  60. Wållberg H et al. (2010) Bi-specific affibody molecules for targeting of EGFR and HER2. Sixth annual protein engineering summit PEGS, May 17–21, Boston. Poster Abstract 78Google Scholar
  61. Weissleder R (2006) Molecular imaging in cancer. Science 312:1168–1171PubMedCrossRefGoogle Scholar
  62. Wikman M, Steffen AC, Gunneriusson E, Tolmachev V, Adams GP, Carlsson J, Ståhl S (2004) Selection and characterization of HER2/neu-binding affibody ligands. Protein Eng Des Sel 17:455–462PubMedCrossRefGoogle Scholar
  63. Wikman M, Rowcliffe E, Friedman M, Henning P, Lindholm L, Olofsson S, Ståhl S (2006) Selection and characterization of an HIV-1 gp120-binding affibody ligand. Biotechnol Appl Biochem 45:93–105PubMedCrossRefGoogle Scholar
  64. Xu L, Aha P, Gu K, Kuimelis RG, Kurz M, Lam T, Lim AC, Liu H, Lohse PA, Sun L et al (2002) Directed evolution of high-affinity antibody mimics using mRNA display. Chem Biol 9:933–942PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  1. 1.Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH)AlbaNova University CenterStockholmSweden
  2. 2.Affibody ABStockholmSweden
  3. 3.Unit of Biomedical Radiations Sciences, Rudbeck LaboratoryUppsala UniversityUppsalaSweden

Personalised recommendations