Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin

Summary

An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [3H]leucine by CEM cells was inhibited by 50% at an M-T151-ricin-A-chain concentration (IC50) of 4.6 nM compared with an IC50 of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC50 values of 20–30 pM. The addition of ricin B chain to CEM cells treated with M-T151—ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly.

Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151—ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [3H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.

This is a preview of subscription content, log in to check access.

References

  1. 1.

    Blakey DC, Wawrzynczak EJ, Wallace PM, Thorpe PE (1988) Antibody-toxin conjugates: a perspective. Prog Allergy 45: 50

  2. 2.

    Cumber AJ, Forrester JA, Foxwell BMJ, Ross WCJ, Thorpe PE (1985) The preparation of antibody-toxin conjugates. Methods Enzymol 112: 207

  3. 3.

    Esworthy RS, Neville DM Jr (1984) A comparative study of ricin and diphtheria toxin-antibody conjugate kinetics on protein synthesis inactivation. J Biol Chem 259: 11 496

  4. 4.

    Forrester JA, McIntosh DP, Cumber AJ, Parnell GD, Ross WCJ (1984) Delivery of ricin and abrin A chains to human carcinoma cells in culture following linkage to monoclonal antibody LICR-LOND-Fib75. Cancer Drug Deliv 1: 283

  5. 5.

    Frankel AE (ed) (1988) Immunotoxins. Kluwer Academic, Boston

  6. 6.

    Fulton RJ, Blakey DC, Knowles PP, Uhr JW, Thorpe PE, Vitetta ES (1986) Purification of ricin A1, A2 and B chains and characterization of their toxicity. J Biol Chem 261: 5314

  7. 7.

    Godal A, Fodstad O, Pihl A (1987) Studies on the mechanism of action of abrin-9.2.27 immunotoxin in human melanoma cell lines. Cancer Res 47: 6243

  8. 8.

    Gregg EO, Bridges SM, York RJ, Longo BL, Houston LL, Glennie MJ, Stevenson FK, Green I (1987) Whole ricin and recombinant ricin A chain idiotype-specific immunotoxins for therapy of guinea pig L2C B cell leukaemia. J Immunol 138: 4502

  9. 9.

    Knowles PP, Thorpe PE (1987) Purification of immunotoxins containing ricin A chain and abrin A chain using Blue Sepharose CL-6B. Anal Biochem 160: 440

  10. 10.

    Leonard JE, Wang Q, Kaplan NO, Royston I (1985) Kinetics of protein synthesis inactivation in human T lymphocytes by selective monoclonal antibody-ricin conjugates. Cancer Res 45: 5263

  11. 11.

    McIntosh DP, Edwards DC, Cumber AJ, Parnell GD, Dean CJ, Ross WCJ, Forrester JA (1983) Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent. FEBS Lett 164: 17

  12. 12.

    Nicolson GL, Blaustein J (1972) The interaction ofRicinus communis agglutinin with normal and tumour cell surfaces. Biochim Biophys Acta 266: 543

  13. 13.

    Preijers FWMB, De Witte T, Rijke-Schilder GPM, Tax WJM, Wessels JMC, Haanen C, Capel PJA (1988) Human T lymphocyte differentiation antigens as target for immunotoxins or complement-mediated cytotoxicity. Scand J Immunol 28: 185

  14. 14.

    Rieber P, Lohmeyer J, Schendel J, Gottlinger J, Brodmann S, Rank G, Heydecke S, Kopp E, Riethmuller G (1984) Characterisation of functional human T cell subsets by monoclonal antibodies. In: Bernard A, Boumsell L, Dausset J, Milstein C, Schlossman SF (eds) Leukocyte typing. Springer-Verlag, New York, p 303

  15. 15.

    Sattentau QJ, Dalgleish AG, Weiss RA, Beverley PCL (1986) Epitopes of the CD4 antigen and HIV infection. Science 234: 1120

  16. 16.

    Street NE, Fulton RJ, Sanders VM, Vitetta ES (1987) Inhibition of the helper function of murine T cells with Fab′-anti-L3T4 ricin A chain immunotoxin. J Immunol 139: 1734

  17. 17.

    Thorpe PE (1985) Antibody carriers of cytotoxic agents in cancer therapy: a review. In: Pinchera A, Doria G, Dammacca F, Bargellesi A (eds) Monoclonal antibodies '84. Biological and clinical applications. Editrice Kurtis, Milan, p 475

  18. 18.

    Thorpe PE, Ross WCJ (1982) The preparation and cytotoxic properties of antibody-toxin conjugates. Immunol Rev 62: 119

  19. 19.

    Thorpe PE, Brown ANF, Foxwell BMJ, Myers C (1983) Blockade of the galactose-binding site of ricin by its linkage to antibody. In: Boss BD, Langman R, Trowbridge I, Dulbecco R (eds) Monoclonal antibodies and cancer. Academic Press, New York, p 117

  20. 20.

    Thorpe PE, Ross WCJ, Brown ANF, Myers CD, Cumber AJ, Foxwell BMJ, Forrester JA (1984) Blockade of the galactose-binding sites of ricin by its linkage to antibody. Eur J Biochem 140: 63

  21. 21.

    Vitetta ES (1986) Synergy between immunotoxins prepared with native ricin A chains and chemically modified ricin B chains. J Immunol 136: 1880

  22. 22.

    Vitetta ES, Cushley W, Uhr JW (1983) Synergy of ricin A chaincontaining immunotoxins and ricin B chain-containing immunotoxins in in vitro killing of neoplastic human B cells. Proc Natl Acad Sci USA 80: 6332

  23. 23.

    Vitetta ES, Fulton RJ, Uhr JW (1984) Cytotoxicity of a cell-reactive immunotoxin containing ricin A chain is potentiated by an anti-immunotoxin containing ricin B chain. J Exp Med 160: 341

  24. 24.

    Vitetta EJ, Fulton RJ, May RD, Till M, Uhr JW (1987) Redesigning nature's poisons to create anti-tumour reagents. Science 238: 1098

  25. 25.

    Wawrzynczak EJ, Thorpe PE (1987) Methods for preparing immunotoxins: effect of the linkage on activity and stability. In: Vogel C-W (ed) Immunoconjugates: antibody conjugates in radioimaging and therapy of cancer. Oxford University Press, New York, p 28

  26. 26.

    Wawrzynczak EJ, Drake AF, Watson GJ, Thorpe PE, Vitetta ES (1988) Ricin B chain-containing immunotoxins prepared with heatdenatured B chain lacking galactose-binding ability potentiate the cytotoxicity of a cell-reactive ricin A chain immunotoxin. Biochim Biophys Acta 971: 55

  27. 27.

    Youle RJ, Neville DM Jr (1982) Kinetics of protein synthesis inactivation by ricin-anti-Thy1.1 monoclonal antibody hybrids. J Biol Chem 257: 1598

  28. 28.

    Youle RJ, Murray GJ, Neville DM Jr (1981) Studies on the galactose-binding site of ricin and the hybrid toxin Man6P-ricin. Cell 23: 551

Download references

Author information

Correspondence to Edward J. Wawrzynczak.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wawrzynczak, E.J., Watson, G.J., Cumber, A.J. et al. Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin. Cancer Immunol Immunother 32, 289–295 (1991). https://doi.org/10.1007/BF01789046

Download citation

Keywords

  • Lactose
  • Leucine
  • Target Antigen
  • Mouse Immunoglobulin
  • Leucine Incorporation