Journal Mobile Options
Table of Contents
Vol. 74, No. 1, 2007
Issue release date: May 2007
Section title: Review
Pathobiology 2007;74:3–14

Bispecific Antibodies: Molecules That Enable Novel Therapeutic Strategies

Fischer N. · Léger O.
NovImmune SA, Geneva, Switzerland
email Corresponding Author

N. Fischer

NovImmune SA

64 av. De la Roseraie

CH–1211 Geneva 4 (Switzerland)

Tel. +41 22 593 5110, Fax +41 22 593 5109, E-Mail


  1. Carter PJ: Potent antibody therapeutics by design. Nat Rev Immunol 2006;6:343–357.
  2. Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495–497.
  3. Hoogenboom HR: Selecting and screening recombinant antibody libraries. Nat Biotechnol 2005;23:1105–1116.
  4. Lonberg N: Human antibodies from transgenic animals. Nat Biotechnol 2005;23:1117–1125.
  5. Cotton RG, Milstein C: Fusion of two immunoglobulin-producing myeloma cells (letter). Nature 1973;244:42–43.
  6. Suresh MR, Cuello AC, Milstein C: Bispecific monoclonal antibodies from hybrid hybridomas. Methods Enzymol 1986;121:210–228.
  7. Suresh MR, Cuello AC, Milstein C: Advantages of bispecific hybridomas in one-step immunocytochemistry and immunoassays. Proc Natl Acad Sci USA 1986;83:7989–7993.
  8. Glennie MJ, McBride HM, Worth AT, Stevenson GT: Preparation and performance of bispecific F(ab’ gamma)2 antibody containing thioether-linked Fab’ gamma fragments. J Immunol 1987;139:2367–2375.
  9. Shalaby MR, Shepard HM, Presta L, Rodrigues ML, Beverley PC, Feldmann M, Carter P: Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. J Exp Med 1992;175:217–225.
  10. Jung G, Freimann U, Von Marschall Z, Reisfeld RA, Wilmanns W: Target cell-induced T cell activation with bi- and trispecific antibody fragments. Eur J Immunol 1991;21:2431–2435.
  11. Pfister DG, Lipton A, Belt R: A phase I trial of the epidermal growth factor receptor-directed bispecific antibody MDX-447 in patients with solid tumors. Proc Am Soc Clin Oncol 1999;18:1667.
  12. Repp R, van Ojik HH, Valerius T, Groenewegen G, Wieland G, Oetzel C, Stockmeyer B, Becker W, Eisenhut M, Steininger H, Deo YM, Blijham GH, Kalden JR, van de Winkel JG, Gramatzki M: Phase I clinical trial of the bispecific antibody MDX-H210 (anti-FcgammaRI × anti-HER-2/neu) in combination with Filgrastim (G-CSF) for treatment of advanced breast cancer. Br J Cancer 2003;89:2234–2243.
  13. Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM, Lee T, Pope SH, Riordan GS, Whitlow M: Single-chain antigen-binding proteins. Science 1988;242:423–426.
  14. Huston JS, Mudgett-Hunter M, Tai MS, Mc Cartney J, Warren F, Haber E, Oppermann H: Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol 1991;203:46–88.
  15. Grosse-Hovest L, Muller S, Minoia R, Wolf E, Zakhartchenko V, Wenigerkind H, Lassnig C, Besenfelder U, Muller M, Lytton SD, Jung G, Brem G: Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing. Proc Natl Acad Sci USA 2004;101:6858–6863.
  16. Kipriyanov SM: Generation and characterization of bispecific tandem diabodies for tumor therapy. Methods Mol Biol 2003;207:323–333.
  17. Korn T, Nettelbeck DM, Volkel T, Muller R, Kontermann RE: 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 2004;6:642–651.
  18. McCall AM, Adams GP, Amoroso AR, Nielsen UB, Zhang L, Horak E, Simmons H, Schier R, Marks JD, Weiner LM: Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis. Mol Immunol 1999;36:433–445.
  19. Holliger P, Prospero T, Winter G: ‘Diabodies’: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci USA 1993;90:6444–6448.
  20. Holliger P, Wing M, Pound JD, Bohlen H, Winter G: Retargeting serum immunoglobulin with bispecific diabodies. Nat Biotechnol 1997;15:632–636.
  21. Perisic O, Webb PA, Holliger P, Winter G, Williams RL: Crystal structure of a diabody, a bivalent antibody fragment. Structure 1994;2:1217–1226.
  22. Poljak RJ: Production and structure of diabodies. Structure 1994;2:1121–1123.
  23. Zhu Z, Zapata G, Shalaby R, Snedecor B, Chen H, Carter P: High level secretion of a humanized bispecific diabody from Escherichia coli. Biotechnology (NY) 1996;14:192–196.
  24. Kipriyanov SM, Moldenhauer G, Schuhmacher J, Cochlovius B, der Lieth CW, Matys ER, Little M: Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics. J Mol Biol 1999;293:41–56.
  25. Volkel T, Korn T, Bach M, Muller R, Kontermann RE: Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies. Protein Eng 2001;14:815–823.
  26. Harris JM, Chess RB: Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2003;2:214–221.
  27. Yang K, Basu A, Wang M, Chintala R, Hsieh MC, Liu S, Hua J, Zhang Z, Zhou J, Li M, Phyu H, Petti G, Mendez M, Janjua H, Peng P, Longley C, Borowski V, Mehlig M, Filpula D: Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation. Protein Eng 2003;16:761–770.
  28. Lu D, Jimenez X, Zhang H, Atkins A, Brennan L, Balderes P, Bohlen P, Witte L, Zhu Z: Di-diabody: a novel tetravalent bispecific antibody molecule by design. J Immunol Methods 2003;279:219–232.
  29. Lu D, Zhang H, Koo H, Tonra J, Balderes P, Prewett M, Corcoran E, Mangalampalli V, Bassi R, Anselma D, Patel D, Kang X, Ludwig DL, Hicklin DJ, Bohlen P, Witte L, Zhu Z: 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 2005;280:19665–19672.
  30. Lu D, Zhang H, Ludwig D, Persaud A, Jimenez X, Burtrum D, Balderes P, Liu M, Bohlen P, Witte L, Zhu Z: Simultaneous blockade of both the epidermal growth factor receptor and the insulin-like growth factor receptor signaling pathways in cancer cells with a fully human recombinant bispecific antibody. J Biol Chem 2004;279:2856–2865.
  31. Pack P, Pluckthun A: Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. Biochemistry 1992;31:1579–1584.
  32. Pack P, Muller K, Zahn R, Pluckthun A: Tetravalent miniantibodies with high avidity assembling in Escherichia coli. J Mol Biol 1995;246:28–34.
  33. Rossi EA, Goldenberg DM, Cardillo TM, McBride WJ, Sharkey RM, Chang CH: Stably tethered multifunctional structures of defined composition made by the dock and lock method for use in cancer targeting. Proc Natl Acad Sci USA 2006;103:6841–6846.
  34. Ridgway JB, Presta LG, Carter P: ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 1996;9:617–621.
  35. Atwell S, Ridgway JB, Wells JA, Carter P: Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J Mol Biol 1997;270:26–35.
  36. Merchant AM, Zhu Z, Yuan JQ, Goddard A, Adams CW, Presta LG, Carter P: An efficient route to human bispecific IgG. Nat Biotechnol 1998;16:677–681.
  37. Ward ES, Gussow D, Griffiths AD, Jones PT, Winter G: Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 1989;341:544–546.
  38. Dooley H, Flajnik MF: Antibody repertoire development in cartilaginous fish. Dev Comp Immunol 2006;30:43–56.
  39. Muyldermans S, Cambillau C, Wyns L: Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci 2001;26:230–235.
  40. Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM: Domain antibodies: proteins for therapy. Trends Biotechnol 2003;21:484–490.
  41. Domantis limited web site: Anti-IL-4/IL-13 dual targeting domain antibody.
  42. Kreutz FT, Wishart DS, Suresh MR: Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography. J Chromatogr B Biomed Sci Appl 1998;714:161–170.
  43. Tarditi L, Camagna M, Parisi A, Vassarotto C, DeMonte LB, Letarte M, Malavasi F, Mariani M: Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies. J Chromatogr 1992;599:13–20.
  44. Bos R, Nieuwenhuizen W: Enhanced transfection of a bacterial plasmid into hybridoma cells by electroporation: application for the selection of hybrid hybridoma (quadroma) cell lines. Hybridoma 1992;11:41–51.
  45. De Lau WB, Van Loon AE, Heije K, Valerio D, Bast BJ: Production of hybrid hybridomas based on HAT(s)-neomycin(r) double mutants. J Immunol Methods 1989;117:1–8.
  46. Karawajew L, Micheel B, Behrsing O, Gaestel M: Bispecific antibody-producing hybrid hybridomas selected by a fluorescence activated cell sorter. J Immunol Methods 1987;96:265–270.
  47. XTLBio web site:
  48. National Institute of Health Clinical Trials web site:
  49. Pritchard KI: Improving outcomes: biologic agents and translational research. 2006 Program and abstracts of the 3rd International BCIRG conference.
  50. Leonard JP, Coleman M, Ketas J, Ashe M, Fiore JM, Furman RR, Niesvizky R, Shore T, Chadburn A, Horne H, Kovacs J, Ding CL, Wegener WA, Horak ID, Goldenberg DM: Combination antibody therapy with epratuzumab and rituximab in relapsed or refractory non-Hodgkin’s lymphoma. J Clin Oncol 2005;23:5044–5051.
  51. Chakravarti A, Chakladar A, Delaney MA, Latham DE, Loeffler JS: The epidermal growth factor receptor pathway mediates resistance to sequential administration of radiation and chemotherapy in primary human glioblastoma cells in a RAS-dependent manner. Cancer Res 2002;62:4307–4315.
  52. Dreier T, Lorenczewski G, Brandl C, Hoffmann P, Syring U, Hanakam F, Kufer P, Riethmuller G, Bargou R, Baeuerle PA: Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody. Int J Cancer 2002;100:690–697.
  53. Helfrich W, Kroesen BJ, Roovers RC, Westers L, Molema G, Hoogenboom HR, de Leij L: Construction and characterization of a bispecific diabody for retargeting T cells to human carcinomas. Int J Cancer 1998;76:232–239.
  54. Korn T, Muller R, Kontermann RE: Bispecific single-chain diabody-mediated killing of endoglin-positive endothelial cells by cytotoxic T lymphocytes. J Immunother 2004;27:99–106.
  55. Ren-Heidenreich L, Davol PA, Kouttab NM, Elfenbein GJ, Lum LG: Redirected T-cell cytotoxicity to epithelial cell adhesion molecule-overexpressing adenocarcinomas by a novel recombinant antibody, E3Bi, in vitro and in an animal model. Cancer 2004;100:1095–1103.
  56. Xiong D, Xu Y, Liu H, Peng H, Shao X, Lai Z, Fan D, Yang M, Han J, Xie Y, Yang C, Zhu Z: Efficient inhibition of human B-cell lymphoma xenografts with an anti-CD20 × anti-CD3 bispecific diabody. Cancer Lett 2002;177:29–39.
  57. Ford CH, Osborne PA, Rego BG, Mathew A: Bispecific antibody targeting of doxorubicin to carcinoembryonic antigen-expressing colon cancer cell lines in vitro and in vivo. Int J Cancer 2001;92:851–855.
  58. Gestin JF, Loussouarn A, Bardies M, Gautherot E, Gruaz-Guyon A, Sai-Maurel C, Barbet J, Curtet C, Chatal JF, Faivre-Chauvet A: Two-step targeting of xenografted colon carcinoma using a bispecific antibody and 188Re-labeled bivalent hapten: biodistribution and dosimetry studies. J Nucl Med 2001;42:146–153.
  59. Kranenborg MH, Boerman OC, Oosterwijk-Wakka JC, de Weijert MC, Corstens FH, Oosterwijk E: Development and characterization of anti-renal cell carcinoma × antichelate bispecific monoclonal antibodies for two-phase targeting of renal cell carcinoma. Cancer Res 1995;55:5864s–5867s.
  60. Bhatia J, Sharma SK, Chester KA, Pedley RB, Boden RW, Read DA, Boxer GM, Michael NP, Begent RH: Catalytic activity of an in vivo tumor targeted anti-CEA scFv::carboxypeptidase G2 fusion protein. Int J Cancer 2000; 85:571–577.
  61. Demanet C, Brissinck J, De Jonge J, Thielemans K: Bispecific antibody-mediated immunotherapy of the BCL1 lymphoma: increased efficacy with multiple injections and CD28-induced costimulation. Blood 1996;87:4390–4398.
  62. Goldstein J, Graziano RF, Sundarapandiyan K, Somasundaram C, Deo YM: Cytolytic and cytostatic properties of an anti-human Fc gammaRI (CD64) × epidermal growth factor bispecific fusion protein. J Immunol 1997;158:872–879.
  63. Heijnen IA, Rijks LJ, Schiel A, Stockmeyer B, van Ojik HH, Dechant M, Valerius T, Keler T, Tutt AL, Glennie MJ, van Royen EA, Capel PJ, van de Winkel JG: Generation of HER-2/neu-specific cytotoxic neutrophils in vivo: efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fcgamma receptor I bispecific antibodies. J Immunol 1997;159:5629–5639.
  64. Kufer P, Lutterbuse R, Baeuerle PA: A revival of bispecific antibodies. Trends Biotechnol 2004;22:238–244.
  65. Offner S, Hofmeister R, Romaniuk A, Kufer P, Baeuerle PA: Induction of regular cytolytic T cell synapses by bispecific single-chain antibody constructs on MHC class I-negative tumor cells. Mol Immunol 2006;43:763–771.
  66. Juweid M, Neumann R, Paik C, Perez-Bacete MJ, Sato J, van Osdol W, Weinstein JN: Micropharmacology of monoclonal antibodies in solid tumors: direct experimental evidence for a binding site barrier. Cancer Res 1992;52:5144–5153.
  67. Saga T, Neumann RD, Heya T, Sato J, Kinuya S, Le N, Paik CH, Weinstein JN: Targeting cancer micrometastases with monoclonal antibodies: a binding-site barrier. Proc Natl Acad Sci USA 1995;92:8999–9003.
  68. Adams GP, Schier R, McCall AM, Simmons HH, Horak EM, Alpaugh RK, Marks JD, Weiner LM: High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules. Cancer Res 2001;61:4750–4755.
  69. Dorvillius M, Garambois V, Pourquier D, Gutowski M, Rouanet P, Mani JC, Pugniere M, Hynes NE, Pelegrin A: Targeting of human breast cancer by a bispecific antibody directed against two tumour-associated antigens: ErbB-2 and carcinoembryonic antigen. Tumour Biol 2002;23:337–347.
  70. Posey JA, Raspet R, Verma U, Deo YM, Keller T, Marshall JL, Hodgson J, Mazumder A, Hawkins MJ: A pilot trial of GM-CSF and MDX-H210 in patients with erbB-2-positive advanced malignancies. J Immunother 1999;22:371–379.
  71. Valone FH, Kaufman PA, Guyre PM, Lewis LD, Memoli V, Ernstoff MS, Wells W, Barth R, Deo Y, Fisher J: Clinical trials of bispecific antibody MDX-210 in women with advanced breast or ovarian cancer that overexpresses HER-2/neu. J Hematother 1995;4:471–475.
  72. Weiner LM, Alpaugh RK, Amoroso AR, Adams GP, Ring DB, Barth MW: Human neutrophil interactions of a bispecific monoclonal antibody targeting tumor and human Fc gamma RIII. Cancer Immunol Immunother 1996;42:141–150.
  73. Cao Y, Lam L: Bispecific antibody conjugates in therapeutics. Adv Drug Deliv Rev 2003;55:171–197.
  74. Kroesen BJ, Buter J, Sleijfer DT, Janssen RA, van der Graaf WT, The TH, de Leij L, Mulder NH: Phase I study of intravenously applied bispecific antibody in renal cell cancer patients receiving subcutaneous interleukin 2. Br J Cancer 1994;70:652–661.
  75. Tibben JG, Boerman OC, Massuger LF, Schijf CP, Claessens RA, Corstens FH: Pharmacokinetics biodistribution and biological effects of intravenously administered bispecific, monoclonal antibody OC/TR F(ab’)2 in ovarian carcinoma patients. Int J Cancer 1996;66:477–483.
  76. Molema G: Design of vascular endothelium-specific drug-targeting strategies for the treatment of cancer. Acta Biochim Pol 2005;52:301–310.
  77. Grosse-Hovest L, Hartlapp I, Marwan W, Brem G, Rammensee HG, Jung G: A recombinant bispecific single-chain antibody induces targeted, supra-agonistic CD28-stimulation and tumor cell killing. Eur J Immunol 2003;33:1334–1340.
  78. Grosse-Hovest L, Wick W, Minoia R, Weller M, Rammensee HG, Brem G, Jung G: 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 2005;117:1060–1064.
  79. Schroder A, Lutterbuse R, Zettl F, Daniel PT, Schwenkenbecker JM, Kufer P, Riethmuller G, Dorken B, Bargou R: A recombinant bispecific single chain antibody CD19 × CD3 induces rapid B cell lymphoma-directed cytotoxicity of unstimulated human T cells (abstract). Blood 1998;92(10 suppl):2101.
  80. Loffler A, Kufer P, Lutterbuse R, Zettl F, Daniel PT, Schwenkenbecher JM, Riethmuller G, Dorken B, Bargou RC: A recombinant bispecific single-chain antibody CD19 × CD3 induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood 2000;95:2098–2103.
  81. Bohlen H, Manzke O, Patel B, Moldenhauer G, Dorken B, von Fliedner V, Diehl V, Tesch H: Cytolysis of leukemic B-cells by T-cells activated via two bispecific antibodies. Cancer Res 1993;53:4310–4314.
  82. Cochlovius B, Kipriyanov SM, Stassar MJ, Christ O, Schuhmacher J, Strauss G, Moldenhauer G, Little M: Treatment of human B cell lymphoma xenografts with a CD3 × CD19 diabody and T cells. J Immunol 2000;165:888–895.
  83. Bargou R, Kufer P, Kirchinger P, Noppeney R, Schuler M, Viardot A, Weigang-Köhler K, Zettl F, Gerecke C, Libicher M, Riethmüller G, Lutterbüse R, Klinger M, Reinhardt C, Baeuerle P: MT103 (anti-CD19 × anti-CD3-BiTE) induces B cell depletion, clearance of bone marrow infiltration and clinical responses in heavily pre-treated NHL patients: first data from dose-escalation study MT103–104. 11th Congress of the European Hematology Association (EHA), Amsterdam, 2006.
  84. Zeidler R, Reisbach G, Wollenberg B, Lang S, Chaubal S, Schmitt B, Lindhofer H: Simultaneous activation of T cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing. J Immunol 1999;163:1246–1252.
  85. Zeidler R, Mysliwietz J, Csanady M, Walz A, Ziegler I, Schmitt B, Wollenberg B, Lindhofer H: The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumour cells. Br J Cancer 2000;83:261–266.
  86. Zeidler R, Mayer A, Gires O, Schmitt B, Mack B, Lindhofer H, Wollenberg B, Walz A: TNFalpha contributes to the antitumor activity of a bispecific trifunctional antibody. Anticancer Res 2001;21:3499–3503.
  87. Heiss MM, Strohlein MA, Jager M, Kimmig R, Burges A, Schoberth A, Jauch KW, Schildberg FW, Lindhofer H: Immunotherapy of malignant ascites with trifunctional antibodies. Int J Cancer 2005;117:435–443.
  88. Stroehlein MA, Lordick F, Ruettinger D, Gruetzner U, Menzel H, Bartelheim K, Jaeger M, Lindhof H, Jauch K, Peschel C, Heiss MM: Treatment of peritoneal carcinomatosis due to GI-tract cancer by intraperitoneal application of the trifunctional antibody catumaxomab (anti-EpcAM × anti-CD3): results of a phase I/II trial (abstract 2529). 2005 ASCO Annual Meeting Proceedings. J Clin Oncol 2005;23/16S:2529.
  89. Ruf P, Lindhofer H: Induction of a long-lasting antitumor immunity by a trifunctional bispecific antibody. Blood 2001;98:2526–2534.
  90. Kiewe P, Hasmuller S, Kahlert S, Heinrigs M, Rack B, Marme A, Korfel A, Jager M, Lindhofer H, Sommer H, Thiel E, Untch M: Phase I trial of the trifunctional anti-HER2 × anti-CD3 antibody ertumaxomab in metastatic breast cancer. Clin Cancer Res 2006;12:3085–3091.
  91. Mirick GR, Bradt BM, Denardo SJ, Denardo GL: A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words. Q J Nucl Med Mol Imaging 2004;48:251–257.
  92. Stein-Streilein J, Hart DA: Effect of route of immunization on development of antibody-forming cells in hilar lymph nodes. Infect Immun 1980;30:391–396.