Generation of Bispecific and Tandem Diabodies

  • Sergey M. Kipriyanov
Part of the Methods in Molecular Biology™ book series (MIMB, volume 178)


A major goal of antibody (Ab)-based tumor targeting has been to specifically deliver a variety of agents, such as radioisotopes, drugs, toxins, lymphokines, and enzymes for imaging and therapy. Intact immunoglobulin G molecules are large (150 kDa) glycoproteins that exhibit a slow systemic clearance, often leading to poor tumor-targeting specificity Fig1 ). Smaller Ab-derived molecules include enzymatically produced 50-kDa Fabs and engineered 25-kDa single-chain Fvs (scFvs) consisting of heavy- and light-chain variable regions (VH and VL) connected by a flexible peptide linker of 14–24 residues (1) ,2; Fig. 2; Fig. 1)). Compared to immunoglobulin G molecules, Fabs and scFvs exhibit significantly improved tumor specificity and intratumoral penetration (3)-5. However, the rapid blood clearance and monovalent nature of these small molecules result in considerably lower quantitative tumor retention (36).


Sucrose Fermentation Lymphoma Electrophoresis Turbidity 


  1. 1.
    Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., et al. (1988) Single-chain antigen-binding proteins. Science 242, 423–426.PubMedCrossRefGoogle Scholar
  2. 2.
    Huston, J. S., Levinson, D., Mudgett Hunter, M., Tai, M. S., Novotny, J., Margolies, M. N., et al. (1988) Protein engineering of antibody binding sites: recovery of specific activity in an antidigoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 5879–5883.CrossRefGoogle Scholar
  3. 3.
    Milenic, D. E., Yokota, T., Filpula, D. R., Finkelman, M. A., Dodd, S. W., Wood, J. F., et al. (1991) Construction, binding properties, metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma monoclonal antibody CC49. Cancer Res. 51, 6363–6371.PubMedGoogle Scholar
  4. 4.
    Yokota, T., Milenic, D. E., Whitlow, M., and Schlom, J. (1992) Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res. 52, 3402–3408.PubMedGoogle Scholar
  5. 5.
    Yokota, T., Milenic, D. E., Whitlow, M., Wood, J. F., Hubert, S. L., and Schlom, J. (1993) Microautoradiographic analysis of the normal organ distribution of radioiodinated single-chain Fv and other immunoglobulin forms. Cancer Res. 53, 3776–3783.PubMedGoogle Scholar
  6. 6.
    Adams, G. P., McCartney, J. E., Tai, M. S., Oppermann, H., Huston, J. S., Stafford, W. F., et al. (1993) Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single-chain Fv. Cancer Res. 53, 4026–4034.PubMedGoogle Scholar
  7. 7.
    Holliger, P., Prospero, T., and Winter, G. (1993) “Diabodies&quote;: small bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. USA 90, 6444–6448.PubMedCrossRefGoogle Scholar
  8. 8.
    Kortt, A. A., Lah, M., Oddie, G. W., Gruen, C. L., Burns, J. E., Pearce, L. A., et al. (1997) Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five-and ten-residue linkers form dimers and with zero-residue linker a trimer. Protein Eng. 10, 423–433.PubMedCrossRefGoogle Scholar
  9. 9.
    Wu, A. M., Chen, W., Raubitschek, A., Williams, L. E., Neumaier, M., Fischer, R., et al. (1996) Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers. Immunotechnology 2, 21–36.PubMedCrossRefGoogle Scholar
  10. 10.
    Adams, G. P., Schier, R., McCall, A. M., Crawford, R. S., Wolf, E. J., Weiner, L. M., and Marks, J. D. (1998) Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu. Br. J. Cancer 77, 1405–1412.PubMedCrossRefGoogle Scholar
  11. 11.
    Holliger, P., Brissinck, J., Williams, R. L., Thielemans, K., and Winter, G. (1996) Specific killing of lymphoma cells by cytotoxic T-cells mediated by a bispecific diabody. Protein Eng. 9, 299–305.PubMedCrossRefGoogle Scholar
  12. 12.
    Kipriyanov, S. M., Moldenhauer, G., Strauss, G., and Little, M. (1998) Bispecific CD3×CD19 diabody for T cell-mediated lysis of malignant human B cells. Int. J. Cancer 77, 763–772.PubMedCrossRefGoogle Scholar
  13. 13.
    Perisic, O., Webb, P. A., Holliger, P., Winter, G., and Williams, R. L. (1994) Crystal structure of a diabody, a bivalent antibody fragment. Structure 2, 1217–1226.PubMedCrossRefGoogle Scholar
  14. 14.
    Zhu, Z., Zapata, G., Shalaby, R., Snedecor, B., Chen, H., and Carter, P. (1996) High level secretion of a humanized bispecific diabody from Escherichia coli. Biotechnology 14, 192–196.Google Scholar
  15. 15.
    Arndt, M. A., Krauss, J., Kipriyanov, S. M., Pfreundschuh, M., and Little, M. (1999) Bispecific diabody that mediates natural killer cell cytotoxicity against xenotransplanted human Hodgkin’s tumors. Blood 94, 2562–2568.PubMedGoogle Scholar
  16. 16.
    Cochlovius, B., Kipriyanov, S. M., Stassar, M. J. J. G., Christ, O., Schuhmacher, J., Strauss, G., Moldenhauer, G., and Little, M. (2000) Treatment of human B cell lymphoma xenografts with a CD3×CD19 diabody and T cells. J. Immunol. 165, 888–895.PubMedGoogle Scholar
  17. 17.
    Ridder, R., Schmitz, R., Legay, F., and Gram, H. (1995) Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris. Biotechnology 13, 255–260.CrossRefGoogle Scholar
  18. 18.
    Shusta, E. V., Raines, R. T., Plückthun, A., and Wittrup, K. D. (1998) Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nature Biotechnol. 16, 773–777.CrossRefGoogle Scholar
  19. 19.
    Kipriyanov, S. M., Moldenhauer, G., Schuhmacher, J., Cochlovius, B., Von der Lieth, C. W., Matys, E. R., and Little, M. (1999) Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics. J. Mol. Biol. 293, 41–56.PubMedCrossRefGoogle Scholar
  20. 20.
    Cochlovius, B., Kipriyanov, S. M., Stassar, M. J. J. G., Schuhmacher, J., Benner, A., Moldenhauer, G., and Little, M. (2000) Cure of Burkitt’s lymphoma in SCID mice by T cells, tetravalent CD3 x CD19 tandem diabody and CD28 costimulation. Cancer Res. 60, 4336–4341.PubMedGoogle Scholar
  21. 21.
    Maurer, R., Meyer, B., and Ptashne, M. (1980) Gene regulation at the right operator (OR) bacteriophage λ. I. OR3 and autogenous negative control by repressor. J. Mol. Biol. 139, 147–161.PubMedCrossRefGoogle Scholar
  22. 22.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  23. 23.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  24. 24.
    Horn, U., Strittmatter, W., Krebber, A., Knupfer, U., Kujau, M., Wenderoth, R., et al. (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, 524–532.PubMedCrossRefGoogle Scholar
  25. 25.
    Kipriyanov, S. M., Moldenhauer, G., and Little, M. (1997) High level production of soluble single chain antibodies in small-scale Escherichia coli cultures. J. Immunol. Methods 200, 69–77.PubMedCrossRefGoogle Scholar
  26. 26.
    Casey, J. L., Keep, P. A., Chester, K. A., Robson, L., Hawkins, R. E., and Begent, R. H. (1995) Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography. J. Immunol. Methods 179, 105–116.PubMedCrossRefGoogle Scholar
  27. 27.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Sergey M. Kipriyanov
    • 1
  1. 1.Affimed Therapeutics AGLadenburgGermany

Personalised recommendations