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Volume 272, Number 44, Issue of October 31, 1997 pp. 27623-27628
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Intracellular Targeting with Low pH-triggered Bispecific Antibodies

(Received for publication, October 24, 1997, and in revised form, August 20, 1997)

From the Boston Biomedical Research Institute, Boston, Massachusetts 02114

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 (t_1/2 = 20 min) indicated that release of CRM107 from the antibody combining site was fast, and its toxic action was unimpeded by this delivery mechanism.

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