Advertisement

Intracellular Targeting Using Bispecific Antibodies

Protocol
  • 609 Downloads
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 25)

Abstract

The technological development and application of bispecific antibodies for biological research have advanced steadily since the idea of creating hybrid reagents with dual specificity was first promulgated by Nisonoff and Rivers (1). It was realized that appropriately designed bispecific antibodies could provide a unique means for selectively delivering biologically active agents onto the surface of target cells so that they could ultimately be internalized (2, 3, 4, 5, 6, 7). Hybrid constructs developed in my laboratory used a specific antibody to reversibly bind the effector molecule within its combining site, whereas the second antibody or ligand component accurately targeted the complex to selected sites on the cell membrane Fig. 1). Those target receptor sites, along with the attached hybrid antibody complex, are subsequently taken inside the cell via receptor-mediated endocytosis. Cytotoxic drugs and toxins were chosen for delivery via the bispecific reagent because the entry of these potent molecules into target cells is signaled by an easily measured intracellular activity (2, 3, 4, 5, 6, 7).
Fig. 1.

Intracellular delivery of effector molecules using bispecific antibodies. A bifunctional carrier is constructed by linking a monoclonal anti-effector antibody to a monoclonal cell-targeting antibody. A noncovalent complex forms when the effector is added and binds to its specific antibody-combining sites. The targeting antibody directs this preformed complex to a distinct receptor site on the cell membrane. Alternatively, cells can be pretreated with the bispecific antibody, allowing the empty combining sites of the cell-bound reagent to be filled by subsequently added effector molecules. Surface-localized complexes quickly enter cells via a receptor-mediated endocytosis pathway. Escape of the effector from the cell vesicle system and passage into the cytosol is achieved but occurs slowly (∼24 h).

Keywords

Diphtheria Toxin Bispecific Antibody Anthrax Toxin 2SO4 Precipitation Hybrid Antibody 
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. 1.
    Nisonoff, A. and Rivers, M. M. (1961) Recombination of a mixture of univalent antibody fragments of different specificity. Arch. Biochem. Biophys. 93, 46CM167.CrossRefGoogle Scholar
  2. 2.
    Raso, V. A. and Griffin, T. (1981) Hybrid antibodies with dual specificity for the delivery of ricin to immunoglobulin-bearing target cells. Cancer Res. 41, 2073–2078.PubMedGoogle Scholar
  3. 3.
    Raso, V. A. (1982) Antibody mediated delivery of toxin molecules to antigen bearing target cells, in Immunological Reviews: Antibody Carriers of Drugs and Toxins in Tumor Therapy, vol. 62 (Moller, G., ed.), Munksgaard, Copenhagen, pp. 93–117.Google Scholar
  4. 4.
    Raso, V. A. and Basala, M. (1984) Monoclonal antibodies as cell targeted carriers of covalently and non-covalently attached toxins, in Receptor Mediated Targeting of Drugs, vol. 82 (Gregoriadis, G., Post, G., Senior, J., and Trouet, A., eds.),NATO Advanced Studies Institute, New York, pp. 119–138.Google Scholar
  5. 5.
    Raso, V. (1994) Immunotargeting intracellular compartments. Anal. Biochem. 222, 297–304.CrossRefPubMedGoogle Scholar
  6. 6.
    Raso, V., Brown, M., and McGrath, J. (1997) Intracellular targeting with low pH triggered bispecific antibodies. J. Biol. Chem. 272, 27,623–27,628.CrossRefPubMedGoogle Scholar
  7. 7.
    Raso, V., Brown, M., McGrath, J., Liu, S., and Stafford, W. (1997) Antibodies capable of releasing diphtheria toxin in response to the low pH found in endosomes. J. Biol. Chem. 272, 27,618–27,622.CrossRefPubMedGoogle Scholar
  8. 8.
    Glennie, M. J., Brennard, D. M., Bryden, F., McBride, H. M., Stirpe, F., Worth, A. T., and Stevenson, G. T. (1988) Bispecific F(AB’)2 antibody for the delivery of saporin in the treatment of lymphoma. J. Immunol. 141, 3662–3670.PubMedGoogle Scholar
  9. 9.
    Sforzini, S., Bolognesi, A., Meazza, R., Marciano, S., Casalini, P., Durkop, H., et al. (1995) Differential sensitivity of CD30+neoplastic cells to gelonin delivered by anti-CD30/anti-gelonin bispecific antibodies. Br. J. Haematol. 90, 572–577.CrossRefPubMedGoogle Scholar
  10. 10.
    Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K. (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes: The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J. Biol. Chem. 262(12), 5908–5912.PubMedGoogle Scholar
  11. 11.
    Geisow, M. L. and Evans, W. H. (1984) pH in the endosome: measurements during pinocytosis and receptor-mediated endocytosis. Exp. Cell Res. 150, 36–46.CrossRefPubMedGoogle Scholar
  12. 12.
    Geisow, M. J. (1984) Fluorescein conjugates as indicators of subcellular pH: a critical evaluation. Exp. Cell Res. 150, 29–35.CrossRefPubMedGoogle Scholar
  13. 13.
    Overly, C. C., Lee, K. D., Berthiaume, E., and Hollenbeck, P. J. (1995) Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine. Proc. Natl. Acad. Sci. USA 92, 3156–3160.CrossRefPubMedGoogle Scholar
  14. 14.
    Nelson, N. (1992) The vacuolar H+-ATPase-one of the most fundamental ion pumps in nature. J. Exp. Biol. 172, 19–27.PubMedGoogle Scholar
  15. 15.
    Blewitt, M. G., Chung, L. A., and London, E. (1985) Effect of pH on the conformation of diphtheria toxin and its implications for membrane penetration. Biochemistry 24, 5458–5464.CrossRefPubMedGoogle Scholar
  16. 16.
    Kennett, R. H. (1980) Monoclonal antibodies, in Fusion Protocols (Kennett, R. H., McKearn, T. J., and Bechtol, K. B., eds.), Plenum Press, New York, pp. 365–367.Google Scholar
  17. 17.
    Choe, S., Bennett, M. J., Fujii, G., Curmi, P. M. G., Kantardjieff, K. A., Collier, R. J., and Eisenberg, D. (1992) The crystal structure of diphtheria toxin. Nature 357, 216–222.CrossRefPubMedGoogle Scholar
  18. 18.
    Sandvig, K. and Olsnes, S. (1980) Diphtheria toxin entry into cells is facilitated by low pH. J. Cell Biol. 87, 828–832.CrossRefPubMedGoogle Scholar
  19. 19.
    Draper, R. K. and Simon, M. I. (1980) The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement. J. Cell Biol. 87, 849–854.CrossRefPubMedGoogle Scholar
  20. 20.
    Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products. J. Biol. Chem. 255, 10,710–10,716.PubMedGoogle Scholar
  21. 21.
    Uchida, T., Pappenheimer, A. M. J., and Greany, R. (1973) Diphtheria toxin and related proteins: I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J. Biol. Chem. 248, 3838–3844.PubMedGoogle Scholar
  22. 22.
    Laird, W. and Groman, N. (1976) Isolation and characterization of tox mutants of corynebacteriophage beta. J. Virol. 19, 220–227.PubMedGoogle Scholar
  23. 23.
    Greenfield, L., Johnson, V. G., and Youle, R. J. (1987) Point mutations in diphtheria toxin separate binding from entry and amplify immunotoxin selectivity. Science 238, 536–539.CrossRefPubMedGoogle Scholar
  24. 24.
    Youle, R. J. (1991) Mutations in diphtheria toxin to improve immunotoxin selectivity and understand toxin entry into cells. Cell Biol. 2, 39–45.Google Scholar
  25. 25.
    Raso, V., unpublished results.Google Scholar
  26. 26.
    Van Renswoude, J., Bridges, K. R., Harford, J. B., and Klausner, R. D. (1982) Receptor-mediated endocytosis of transferrin and the uptake of Fe in K562 cells: Identification of a nonlysosomal acidic compartment. Proc. Natl. Acad. Sci. USA 79, 6186–6190.CrossRefPubMedGoogle Scholar
  27. 27.
    Kim, K. and Groman, N. B. (1965) In vitro inhibition of diphtheria toxin action by ammonium salts and amines. J. Bacteriol. 90, 1552–1556.PubMedGoogle Scholar
  28. 28.
    Brennan, M., Davison, P. F., and Paulus, H. (1985) Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. Science 228, 81–83.CrossRefGoogle Scholar
  29. 29.
    Friedlander, A. M. (1986) Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J. Biol. Chem. 261, 7123–7126.PubMedGoogle Scholar
  30. 30.
    Milne, J. C., Furlong, D., Hanna, P. C., Wall, J. S., and Collier, R. J. (1994) Anthrax protective antigen forms oligomers during intoxication of mammalian cells. J. Biol. Chem. 269, 20,607–20,612.PubMedGoogle Scholar
  31. 31.
    Carroll, S. F., Barbieri, J. T., and Collier, R. J. (1988) Diphtheria toxin: purification and properties. Meth. Enzymol. 165, 68–76.CrossRefPubMedGoogle Scholar
  32. 32.
    Reale, F. R., Griffin, T. W., Compton, J. M., Graham, S., Townes, P. L., and Bogden, A. (1987) Characterization of a human malignant mesothelioma cell line (H-MESO-1): A biphasic solid and ascitic tumor model. Cancer Res. 47, 3199–3205.PubMedGoogle Scholar
  33. 33.
    Griffin, T. W., Richardson, C., Houston, L. L., LePage, D., Bogden, A., and Raso, V. (1987) Antitumor activity of intraperitoneal immunotoxins in a nude mouse model of human malignant mesothelioma. Cancer Res. 47, 4266–4270.PubMedGoogle Scholar
  34. 34.
    Griffin, T., Rybak, M. E., Recht, L., Singh, M., Salimi, A., and Raso, V. A. (1993) Potentiation of antitumor immunotoxins by liposomal monensin. J. Natl. Cancer Inst. 85, 292–298.CrossRefPubMedGoogle Scholar
  35. 35.
    Rappuoli, R., Perugini, M., Marsili, I., and Fabbiani, S. (1983) Rapid purification of diphtheria toxin by phenyl sepharose and DEAE-cellulose chromatography. J. Chromatog. 268, 543–548.CrossRefGoogle Scholar
  36. 36.
    Fracker, P. J. and Speck, J. C. J. (1978) Protein and cell membrane iodinations with sparingly soluble chloroamide, 1,3,4,6,-tetrachloro-3a,6a-diphenylglycoluril. Biochem. Biophys. Res. Comm. 80, 849–857.CrossRefGoogle Scholar
  37. 37.
    Hayakawa, S., Uchida, T., Mekada, E., Moynihan, M. R., and Okada, Y. (1983) Monoclonal antibody against diphtheria toxin: effect on toxin binding and entry into cells. J. Biol. Chem. 258(7), 4311–4317.PubMedGoogle Scholar
  38. 38.
    Zucker, D. R. and Murphy, J. R. (1984) Monoclonal antibody analysis of diphtheria toxin-I. Localization of epitopes and neutralization of cytotoxicity. Mol. Immunol. 21, 785–793.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  1. 1.Boston Biomedical Research InstituteBoston

Personalised recommendations