Generation and Characterization of Bispecific Tandem Diabodies for Tumor Therapy

  • Sergey M. Kipriyanov
Part of the Methods in Molecular Biology™ book series (MIMB, volume 207)


Bispecific antibodies (BsAb) provide an effective means of retargeting cytotoxic effector cells against tumor cells (1). They have mainly been produced using murine hybrid hybridomas (2) or by chemical crosslinking (3,4). However, the immunogenicity of BsAb derived from rodent monoclonal antibodies (MAbs) is a major drawback for clinical use (5). They are also difficult to produce and purify in large quantities. Recent advances in recombinant antibody technology have provided several alternative methods for constructing and producing BsAb molecules (6) (Fig. 1). For example, single chain Fv (scFv) fragments have been genetically fused with adhesive polypeptides (7) or protein domains (8) to facilitate the formation of heterodimers (Fig. 1A). The genetic engineering of scFv-scFv tandems linked with a third polypeptide linker has also been carried out in several laboratories (9,10) (Fig. 1B). A bispecific diabody was obtained by the non-covalent association of two single chain fusion products consisting of the VH domain from one antibody connected by a short linker to the VL domain of another antibody (11,12) (see  Chapter 18 of Arndt and Krauss in this issue) (Fig. 1C). The two antigen binding domains have been shown by crystallographic analysis to be on opposite sides of the diabody such that they are able to crosslink two cells (13). However, the co-secretion of two hybrid scFv fragments can give rise to two types of dimer: active heterodimers and inactive homodimers. A second problem is that the two chains of diabodies are held together by noncovalent associations of the VH and VL domains and can diffuse away from one another. Moreover, to ensure the assembly of a functional diabody, both hybrid scFv fragments must be expressed in the same cell in similar amounts. This latter requirement is difficult to uphold in eukaryotic expression systems such as yeast, which are often preferred because high yields of enriched product can be obtained (14,15). Finally, the small size of bispecific diabodies (50–60 kDa) leads to their rapid clearance from the bloodstream through the kidneys, thus requiring the application of relatively high doses for therapy. In contrast to native antibodies, all of the aforementioned bispecific molecules have only one binding domain for each specificity. However, bivalent binding is an important means of increasing the functional affinity and possibly the selectivity for particular cell types carrying densely clustered antigens.
Fig. 1.

Schematic representation of Fv-based bispecific recombinant antibody constructs and their genes. (A) Bispecific miniantibody assembled from dimerization cassettes based either on leucine zippers (Jun and Fos) (7) or on the antibody first constant domains (CH1 and CL) (8). In this case, two scFv antibody fragments of different specificities (A and B) are fused to adhesive self-associating peptide or protein domains (AD). (B) Bispecific scFv-scFv tandem [(scFv)2] and four-domain gene construct for its production. (C) Bispecific diabody formed by non-covalent association of two hybrid scFv fragments consisting of VH and VL domains of different specificities. (D) Single chain fourdomain gene construct for production of dimeric or tetrameric bispecific molecules. Depending on the linker length, either single-chain diabody (left), or tetravalent tandem diabody (right) can be formed. Antibody variable domains (VH, VL), peptide linkers (L) and antigen-binding sites (A or B) of Fv modules are indicated. The locations of promoter/operator (p/o), ribosome binding sites (rbs), and signal peptides for secretion in bacteria (SP) are also shown.


Antibody Fragment Bispecific Antibody Start Buffer Antigen Binding Domain Noncovalent Association 
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.


  1. 1.
    Fanger M. W., Morganelli P. M., and Guyre P. M. (1992). Bispecific antibodies. Crit. Rev. Immunol. 12, 101–124.PubMedGoogle Scholar
  2. 2.
    Bohlen H., Hopff T., Manzke O., Engert A., Kube D., Wickramanayake P. D., et al. (1993). Lysis of malignant B cells from patients with B-chronic lymphocytic leukemia by autologous T cells activated with CD3 × CD19 bispecific antibodies in combination with bivalent CD28 antibodies. Blood 82, 1803–1812.PubMedGoogle Scholar
  3. 3.
    Brennan M., Davidson P. F., and Paulus H. (1985). Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. Science 229, 81–83.PubMedCrossRefGoogle Scholar
  4. 4.
    Glennie M. J., McBride H. M., Worth A. T., and Stevenson G. T. (1987). Preparation and performance of bispecific F(ab′γ)2 antibody containing thioether-linked Fab′γ fragments. J. Immunol. 139, 2367–2375.PubMedGoogle Scholar
  5. 5.
    Khazaeli M. B., Conry R. M., and LoBuglio A. F. (1994). Human immune response to monoclonal antibodies. J. Immunother. 15, 42–52.CrossRefGoogle Scholar
  6. 6.
    Plückthun A. and Pack P. (1997). New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology 3, 83–105.PubMedCrossRefGoogle Scholar
  7. 7.
    de Kruif J. and Logtenberg T. (1996). Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library. J. Biol. Chem. 271, 7630–7634.PubMedCrossRefGoogle Scholar
  8. 8.
    Müller K. M., Arndt K. M., Strittmatter W., and Plückthun A. (1998). The first constant domain (CH1 and CL) of an antibody used as heterodimerization domain for bispecific miniantibodies. FEBS Lett. 422, 259–264.PubMedCrossRefGoogle Scholar
  9. 9.
    Gruber M., Schodin B. A., Wilson E. R., and Kranz D. M. (1994). Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli. J. Immunol. 152, 5368–5374.PubMedGoogle Scholar
  10. 10.
    Kurucz I., Titus J. A., Jost C. R., Jacobus C. M., and Segal D. M. (1995). Retargeting of CTL by an efficiently refolded bispecific single-chain Fv dimer produced in bacteria. J. Immunol. 154, 4576–4582.PubMedGoogle Scholar
  11. 11.
    Holliger P., Prospero T., and Winter G. (1993). “Diabodies”: small bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. USA 90, 6444–6448.PubMedCrossRefGoogle Scholar
  12. 12.
    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
  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.
    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.PubMedCrossRefGoogle Scholar
  15. 15.
    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. Nat. Biotechnol. 16, 773–777.PubMedCrossRefGoogle Scholar
  16. 16.
    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
  17. 17.
    Cochlovius B., Kipriyanov S. M., Stassar M. J., Schuhmacher J., Benner A., Moldenhauer G., and Little M. (2000). Cure of Burkitt’s lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 × CD19 tandem diabody, and CD28 costimulation. Cancer Res. 60, 4336–4341.PubMedGoogle Scholar
  18. 18.
    Maurer R., Meyer B., and Ptashne M. (1980). Gene regulation at the right operator (O R) bacteriophage λ. I. OR3 and autogenous negative control by repressor. J. Mol. Biol. 139, 147–161.PubMedCrossRefGoogle Scholar
  19. 19.
    Sambrook J., Fritsch E. F., and Maniatis T. (1989}) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  20. 20.
    Laemmli U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  21. 21.
    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
  22. 22.
    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
  23. 23.
    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
  24. 24.
    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. 2003

Authors and Affiliations

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

Personalised recommendations