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Comparison of protein synthesis inhibition kinetics and cell killing induced by immunotoxins
1Chemical Engineering Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Immunotoxins comprised of a monoclonal antibody covalently coupled to recombinant ricin A chain or to a binding-defective form of diphtheria toxin were compared with respect to their rates of protein synthesis inhibition and efficiencies of killing target cells. Protein synthesis inhibition rates were established by measuring the incorporation of L-[14C]leucine in toxin-treated cells relative to untreated cells at several times after exposure of cells to an immunotoxin. Cell killing was assessed by a limiting dilution assay which measures the number of cells surviving toxin treatment relative to untreated cells. At equivalent protein concentrations, the diphtheria toxin immunotoxin inhibited protein synthesis significantly more rapidly than the ricin A immunotoxin but, contrary to previous predictions, achieved a significantly lower cell kill. Thus, the kinetics of protein synthesis inactivation do not necessarily correlate with killing efficiencies. Possible explanations for these results are that the effect of the diphtheria toxin immunotoxin on protein synthesis is partially reversible or that the diphtheria toxin immunotoxin enters the cytosol at a faster rate than the ricin A immunotoxin but also is degraded at a faster rate.
Insights
Diphtheria toxin immunotoxins inhibit protein synthesis faster than ricin A immunotoxins, but show lower cell kill efficiencies. Protein synthesis inhibition kinetics do not always correlate with immunotoxin cell killing efficacy.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Immunotoxins combine antibodies with cytotoxic proteins to target specific cells.
- Ricin A chain and diphtheria toxin are potent protein synthesis inhibitors used in immunotoxin development.
Purpose of the Study:
- To compare the protein synthesis inhibition rates and cell killing efficiencies of immunotoxins based on ricin A chain versus diphtheria toxin.
- To investigate the correlation between the kinetics of protein synthesis inhibition and cell killing efficacy.
Main Methods:
- Immunotoxins were synthesized by covalently coupling monoclonal antibodies to either recombinant ricin A chain or a binding-defective diphtheria toxin.
- Protein synthesis inhibition was measured by L-[14C]leucine incorporation in treated versus untreated cells over time.
- Cell killing efficiency was assessed using a limiting dilution assay to determine surviving cell numbers.
Main Results:
- At equivalent protein concentrations, diphtheria toxin immunotoxins inhibited protein synthesis significantly faster than ricin A immunotoxins.
- Contrary to predictions, diphtheria toxin immunotoxins achieved significantly lower cell kill efficiencies compared to ricin A immunotoxins.
- The rate of protein synthesis inactivation did not directly correlate with the overall cell killing efficiency.
Conclusions:
- The kinetics of protein synthesis inhibition by immunotoxins do not necessarily predict their cell killing efficacy.
- Potential explanations for the observed discrepancy include partial reversibility of diphtheria toxin effects or differential rates of cytosol entry and degradation.