• Jonas V. Schaefer
  • Peter Lindner
  • Andreas PlückthunEmail author
Part of the Springer Protocols Handbooks book series (SPH)


Recombinant antibodies have become a standard component of research, diagnostics, and therapy. In the development of recombinant antibodies – irrespective of the final format – a monovalent construct is virtually always the first protein to be tested. This is due to the fact that essentially all selection systems use such formats, and that the periplasmic production of scFv and Fab fragments has now become standard. Nonetheless, some tasks require an increase of the avidity to the respective antigens, antibody, as well as the fragment size. A convenient way to rather quickly achieve both is by fusing a hinge region followed by a dimerizing or oligomerizing structure to the C-terminus of the antibody fragment, creating the so-called “miniantibodies”. Compared to other available bivalent or bispecific formats, miniantibodies distinguish themselves by their rotational freedom and flexibility, being similar to full-length antibodies. This protocol describes the modular conversion of scFv fragments into miniantibodies, resulting in a final multimerized structure with two or four binding sites, and we present different self-associating domains suitable for this task. Additionally, we also provide information on their production and discuss how to improve the yield of soluble antibody fragments.


Antibody Fragment Oligomerization Domain Soluble Aggregate scFv Fragment Dimerization Motif 
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.





Phosphate buffered saline


Single-chain Fv fragment




Luria-Bertani media


Super broth media



This chapter is based on the original work of Peter Pack, Jörg Willuda and Susanne Kubetzko, with subsequent contributions from Kerstin Blank and Barbara Klinger.


  1. Arndt KM, Pelletier JN, Müller KM, Alber T, Michnick SW, Plückthun A (2000) A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble. J Mol Biol 295:627–639PubMedCrossRefGoogle Scholar
  2. Arndt KM, Müller KM, Plückthun A (2001) Helix-stabilized Fv (hsFv) antibody fragments: substituting the constant domains of a Fab fragment for a heterodimeric coiled-coil domain. J Mol Biol 312:221–228PubMedCrossRefGoogle Scholar
  3. Bass S, Gu Q, Christen A (1996) Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA. J Bacteriol 178:1154–1161PubMedGoogle Scholar
  4. Bothmann H, Plückthun A (1998) Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nat Biotechnol 16:376–380PubMedCrossRefGoogle Scholar
  5. Bothmann H, Plückthun A (2000) The periplasmic Escherichia coli peptidylprolyl cis, trans-isomerase FkpA: I. Increased functional expression of antibody fragments with and without cis-prolines. J Biol Chem 275:17100–17105PubMedCrossRefGoogle Scholar
  6. Crothers DM, Metzger H (1972) The influence of polyvalency on the binding properties of antibodies. Immunochemistry 9:341–357PubMedCrossRefGoogle Scholar
  7. Deyev SM, Waibel R, Lebedenko EN, Schubiger AP, Plückthun A (2003) Design of multivalent complexes using the barnase*barstar module. Nat Biotechnol 21:1486–1492PubMedCrossRefGoogle Scholar
  8. Dürr E, Jelesarov I, Bosshard HR (1999) Extremely fast folding of a very stable leucine zipper with a strengthened hydrophobic core and lacking electrostatic interactions between helices. Biochemistry 38:870–880PubMedCrossRefGoogle Scholar
  9. Eisenberg D, Wilcox W, Eshita SM, Pryciak PM, Ho SP, DeGrado WF (1986) The design, synthesis, and crystallization of an alpha-helical peptide. Proteins 1:16–22PubMedCrossRefGoogle Scholar
  10. Ewert S, Honegger A, Plückthun A (2004) Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering. Methods 34:184–199PubMedCrossRefGoogle Scholar
  11. Ge L, Knappik A, Pack P, Freund C, Plückthun A (1995) Expressing antibodies in Escherichia coli. In: Borrebaeck C (ed) Antibody engineering, 2nd ed. Oxford University Press, London, pp 229–236Google Scholar
  12. Harbury PB, Zhang T, Kim PS, Alber T (1993) A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science 262:1401–1407PubMedCrossRefGoogle Scholar
  13. Hill RB, Degrado WF (1998) Solution structure of alpha-2D, a native-like de novo designed protein. J Am Chem Soc 120:1138–1145CrossRefGoogle Scholar
  14. Holliger P, Prospero T, Winter G (1993) “Diabodies”: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci USA 90:6444–6448PubMedCrossRefGoogle Scholar
  15. Honegger A, Malebranche AD, Röthlisberger D, Plückthun A (2009) The influence of the framework core residues on the biophysical properties of immunoglobulin heavy chain variable domains. Protein Eng Des Sel 22:121–134PubMedCrossRefGoogle Scholar
  16. Horn U, Strittmatter W, Krebber A, Knüpfer U, Kujau M, Wenderoth R, Müller K, Matzku S, Plückthun A, Riesenberg D (1996) High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions. Appl Microbiol Biotechnol 46:524–532PubMedCrossRefGoogle Scholar
  17. Hu S, Shively L, Raubitschek A, Sherman M, Williams LE, Wong JY, Shively JE, Wu AM (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
  18. Huston JS, Mudgett-Hunter M, Tai MS, McCartney J, Warren F, Haber E, Oppermann H (1991) Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol 203:46–88PubMedCrossRefGoogle Scholar
  19. Jeffrey PD, Gorina S, Pavletich NP (1995) Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 Ångstroms. Science 267:1498–1502PubMedCrossRefGoogle Scholar
  20. Jung S, Plückthun A (1997) Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. Protein Eng 10:959–966PubMedCrossRefGoogle Scholar
  21. Kaufmann M, Lindner P, Honegger A, Blank K, Tschopp M, Capitani G, Plückthun A, Grütter MG (2002) Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen. J Mol Biol 318:135–147PubMedCrossRefGoogle Scholar
  22. Kellner C, Bruenke J, Stieglmaier J, Schwemmlein M, Schwenkert M, Singer H, Mentz K, Peipp M, Lang P, Oduncu F, Stockmeyer B, Fey GH (2008) A novel CD19-directed recombinant bispecific antibody derivative with enhanced immune effector functions for human leukemic cells. J Immunother 31:871–884PubMedCrossRefGoogle Scholar
  23. Kipriyanov SM, Moldenhauer G, Schuhmacher J, Cochlovius B, Von der Lieth CW, Matys ER, Little M (1999) Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics. J Mol Biol 293:41–56PubMedCrossRefGoogle Scholar
  24. Knappik A, Plückthun A (1994) An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments. Biotechniques 17:754–761PubMedGoogle Scholar
  25. Knappik A, Plückthun A (1995) Engineered turns of a recombinant antibody improve its in vivo folding. Protein Eng 8:81–89PubMedCrossRefGoogle Scholar
  26. Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun A (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods 201:35–55PubMedCrossRefGoogle Scholar
  27. Kubetzko S, Balic E, Waibel R, Zangemeister-Wittke U, Plückthun A (2006) PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting. J Biol Chem 281:35186–35201PubMedCrossRefGoogle Scholar
  28. Kügler M, Stein C, Schwenkert M, Saul D, Vockentanz L, Huber T, Wetzel SK, Scholz O, Plückthun A, Honegger A, Fey GH (2009) Stabilization and humanization of a single-chain Fv antibody fragment specific for human lymphocyte antigen CD19 by designed point mutations and CDR-grafting onto a human framework. Protein Eng Des Sel 22:135–147PubMedCrossRefGoogle Scholar
  29. Lindner P, Plückthun A (2001) Miniantibodies. In: Kontermann R, Dübel S (eds) Antibody engineering. Springer, Berlin, pp 637–647Google Scholar
  30. Lindner P, Guth B, Wülfing C, Krebber C, Steipe B, Müller F, Plückthun A (1992) Purification of native proteins from the cytoplasm and periplasm of Escherichia coli using IMAC and histidine tails: a comparison of proteins and protocols. Methods 4:41–56CrossRefGoogle Scholar
  31. Lindner P, Bauer K, Krebber A, Nieba L, Kremmer E, Krebber C, Honegger A, Klinger B, Mocikat R, Plückthun A (1997) Specific detection of his-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions. Biotechniques 22:140–149PubMedGoogle Scholar
  32. Maurer R, Meyer B, Ptashne M (1980) Gene regulation at the right operator (OR) bacteriophage lambda. I. OR3 and autogenous negative control by repressor. J Mol Biol 139:147–161PubMedCrossRefGoogle Scholar
  33. Mittl PR, Chene P, Grütter MG (1998) Crystallization and structure solution of p53 (residues 326–356) by molecular replacement using an NMR model as template. Acta Crystallogr D Biol Crystallogr 54:86–89PubMedCrossRefGoogle Scholar
  34. Müller KM, Arndt KM, Plückthun A (1998a) A dimeric bispecific miniantibody combines two specificities with avidity. FEBS Lett 432:45–49PubMedCrossRefGoogle Scholar
  35. Müller KM, Arndt KM, Plückthun A (1998b) Model and simulation of multivalent binding to fixed ligands. Anal Biochem 261:149–158PubMedCrossRefGoogle Scholar
  36. Müller KM, Arndt KM, Strittmatter W, Plückthun A (1998c) The first constant domain (CH1 and CL) of an antibody used as heterodimerization domain for bispecific miniantibodies. FEBS Lett 422:259–264PubMedCrossRefGoogle Scholar
  37. Nieba L, Honegger A, Krebber C, Plückthun A (1997) Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng 10:435–444PubMedCrossRefGoogle Scholar
  38. O'Shea EK, Klemm JD, Kim PS, Alber T (1991) X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254:539–544PubMedCrossRefGoogle Scholar
  39. Pack P, Plückthun A (1992) Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric Fv fragments with high avidity in Escherichia coli. Biochemistry 31:1579–1584PubMedCrossRefGoogle Scholar
  40. Pack P, Kujau M, Schroeckh V, Knüpfer U, Wenderoth R, Riesenberg D, Plückthun A (1993) Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli. Biotechnology (NY) 11:1271–1277Google Scholar
  41. Pack P, Müller K, Zahn R, Plückthun A (1995) Tetravalent miniantibodies with high avidity assembling in Escherichia coli. J Mol Biol 246:28–34PubMedCrossRefGoogle Scholar
  42. Plückthun A, Pack P (1997) New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology 3:83–105PubMedCrossRefGoogle Scholar
  43. Plückthun A, Krebber A, Krebber C, Horn U, Knüpfer U, Wenderoth R, Nieba L, Proba K, Riesenberg D (1996) Producing antibodies in Escherichia coli: Fom PCR to fermentation. In: McCafferty J, Hoogenboom H (eds) Antibody engineering: a practical approach. IRL Press, Oxford, pp 203–252Google Scholar
  44. Rheinnecker M, Hardt C, Ilag LL, Kufer P, Gruber R, Hoess A, Lupas A, Rottenberger C, Plückthun A, Pack P (1996) Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen. J Immunol 157:2989–2997PubMedGoogle Scholar
  45. Rudolph R, Lilie H (1996) In vitro folding of inclusion body proteins. FASEB J 10:49–56PubMedGoogle Scholar
  46. Schroeckh V, Kujau M, Knüpfer U, Wenderoth R, Mörbe J, Riesenberg D (1996) Formation of recombinant proteins in Escherichia coli under control of a nitrogen regulated promoter at low and high cell densities. J Biotechnol 49:45–58PubMedCrossRefGoogle Scholar
  47. Todorovska A, Roovers RC, Dolezal O, Kortt AA, Hoogenboom HR, Hudson PJ (2001) Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J Immunol Methods 248:47–66PubMedCrossRefGoogle Scholar
  48. Willuda J, Honegger A, Waibel R, Schubiger PA, Stahel R, Zangemeister-Wittke U, Pluckthun A (1999) High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Res 59:5758–5767PubMedGoogle Scholar
  49. Willuda J, Kubetzko S, Waibel R, Schubiger PA, Zangemeister-Wittke U, Plückthun A (2001) Tumor targeting of mono-, di-, and tetravalent anti-p185(HER-2) miniantibodies multimerized by self-associating peptides. J Biol Chem 276:14385–14392PubMedGoogle Scholar
  50. Woolfson DN (2005) The design of coiled-coil structures and assemblies. Adv Protein Chem 70:79–112PubMedCrossRefGoogle Scholar
  51. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119PubMedCrossRefGoogle Scholar
  52. Zhang J, Tanha J, Hirama T, Khieu NH, To R, Tong-Sevinc H, Stone E, Brisson JR, MacKenzie CR (2004) Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents. J Mol Biol 335:49–56PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Jonas V. Schaefer
    • 1
  • Peter Lindner
    • 1
  • Andreas Plückthun
    • 1
    Email author
  1. 1.Biochemisches InstitutUniversität ZürichZürichSwitzerland

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