Abstract
Bispecific antibodies were designed to deliver a reversibly bound ligand into target cells and then spontaneously release it upon passage into acidified vesicles. These reagents were assembled by coupling monoclonal antibodies that recognize acid-sensitive epitopes on diphtheria toxin to cell type-specific monoclonal antibodies. The dual binding capacity of the bispecific antibodies was confirmed by delivery of 125I-diphtheria toxin to target molecules present on intact cells. Bispecific antibodies directed against transferrin receptors on human cells were loaded with toxin and tested for cytotoxicity. The mutant diphtheria toxins CRM107 and CRM45 were used since their inability to bind cell receptors renders them ordinarily nontoxic. Their full cytotoxic potential, however, was restored via bispecific antibody-mediated delivery and release within low pH intracellular vesicles. Cytotoxicity was shown to be specific by blocking receptor sites and to be acidification-dependent by protection using NH4Cl to raise endosomal pH. Kinetics for inhibition of cellular protein synthesis was identical for native diphtheria toxin and the bispecific antibody. CRM107 combination. The rate of inhibition (t1/2 = 20 min) indicated that release of CRM107 from the antibody combining site was fast, and its toxic action was unimpeded by this delivery mechanism.
Highlights
Diphtheria toxin acts via a well defined intracellular pathway that culminates with an obligatory transfer of its toxic moiety from acidic endosomes to the cytosol [6, 7]
Dual specificity was verified by measuring the capacity of these reagents to attach simultaneously to transferrin receptors on the cell and bind 125I-toxin
Whereas CRM107 alone is incapable of entering cells and inhibiting protein synthesis, it became a very effective and rapid-acting cytotoxin when used in combination with the anti-transferrin receptor/anti-DT bispecific antibody, 7D3/5E8
Summary
Materials—Na[125I] (17 Ci/mg) and L-[3,4,5-3H]leucine (140 Ci/mmol) were purchased from NEN Life Science Products. To produce an appropriate bispecific antibody combination, the SPDP-substituted anti-transferrin receptor antibody was reduced with 50 mM dithiothreitol for 30 min, and the protein fraction was isolated by passage through a Sephadex G-25 column equilibrated with PBS. This thiolated antibody was mixed with an SPDP-substituted anti-DT antibody, and the com-. The bispecific antibody and CRM toxins were added either separately or mixed first so that the specified final concentration was achieved by diluting the preformed complex in the microtiter well Both methods of treatment gave essentially identical results. The coefficient of variation of the assay was usually Ͻ 0.1
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