Related Experiment Videos
Mutations in diphtheria toxin separate binding from entry and amplify immunotoxin selectivity
L Greenfield1, V G Johnson, R J Youle
1Department of Microbial Genetics, Cetus Corporation, Emeryville, CA 94608.
Abstract:
Monoclonal antibodies linked to toxic proteins (immunotoxins) can selectively kill some tumor cells in vitro and in vivo. However, reagents that combine the full potency of the native toxins with the high degree of cell type selectivity of monoclonal antibodies have not previously been designed. Two heretofore inseparable activities on one polypeptide chain of diphtheria toxin and ricin account for the failure to construct optimal reagents. The B chains (i) facilitate entry of the A chain to the cytosol, which allows immunotoxins to efficiently kill target cells, and (ii) bind to receptors present on most cells, which imparts to immunotoxins a large degree of non-target cell toxicity. This report identifies point mutations in the B polypeptide chain of diphtheria toxin that block binding but allow cytosol entry. Three mutants of diphtheria toxin have 1/1,000 to 1/10,000 the toxicity and 1/100 to 1/8,000 the binding activity of diphtheria toxin. Linking of either of two of the inactivated mutant toxins (CRM103, Phe508; CRM107, Phe390, Phe525) to a monoclonal antibody specific for human T cells reconstitutes full target-cell toxicity--indistinguishable from that of the native toxin linked to the same antibody--without restoring non-target cell toxicity. This separation of the entry function from the binding function generates a uniquely potent and cell type-specific immunotoxin that retains full diphtheria toxin toxicity, yet is four to five orders of magnitude less toxic than the native toxin is to nontarget cells.
Insights
Engineered immunotoxins combine potent cancer cell killing with high specificity. Modified diphtheria toxin B chains enable targeted delivery and toxicity without harming non-target cells, improving cancer therapy potential.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Immunotoxins, combining monoclonal antibodies with toxic proteins, aim to selectively eliminate tumor cells.
- Previous attempts to create potent and specific immunotoxins were hindered by inseparable toxin functions.
- Diphtheria toxin's B chain facilitates cell entry and binds to non-target cell receptors, causing toxicity.
Purpose of the Study:
- To design novel immunotoxins with enhanced cell-type specificity and retained potency.
- To separate the cell entry function from the receptor binding function of diphtheria toxin.
Main Methods:
- Point mutations were introduced into the B polypeptide chain of diphtheria toxin.
- Mutant diphtheria toxins were assessed for toxicity and binding activity.
- Mutant toxins were linked to a monoclonal antibody targeting human T cells.
Main Results:
- Mutant diphtheria toxins showed significantly reduced toxicity and binding affinity.
- Immunotoxins created with inactivated mutants retained full target-cell toxicity.
- Non-target cell toxicity was substantially reduced in the engineered immunotoxins.
Conclusions:
- Separating diphtheria toxin's entry and binding functions yields potent, cell-specific immunotoxins.
- This approach significantly reduces toxicity to non-target cells while maintaining efficacy.
- These findings offer a promising strategy for developing safer and more effective immunotoxin therapies.