Vic Raso

Monensin is obligatory for the cytotoxic action of a disulfide linked methotrexate-anti-transferrin receptor conjugate

Methotrexate (MTX) in the form of a gamma-cysteinylglycine derivative was disulfide linked to a monoclonal antibody reactive with the human transferrin receptor to give an antibody-MTX conjugate (anti-TfR-MTX). Antibody directed delivery of MTX to cell surface receptors was readily detected by flow cytometry using an anti-MTX antibody plus a secondary fluorescent antibody probe. Despite the presence of ample drug on the cell membrane, the conjugate alone was not cytotoxic over the course of several days. Expression of specific toxic activity, however, was obtained in conjunction with the carboxylic ionophore monensin, in whose presence anti-TfR-MTX displayed an IC50 of 8 X 10(-8) M. These results suggest that the ionophore causes antibody-drug conjugate to bypass the normal transferrin receptor cyclic pathway, allowing sufficient drug to reach, bind to, and inactivate intracellular dihydrofolate reductase.

Monoclonal Antibodies as Cell Targeted Carriers of Covalently and Non-Covalently Attached Toxins

The strategy of using cell specific monoclonal antibodies to modify the indiscriminate binding characteristics of lethal toxins has stimulated considerable interest because of its therapeutic potential. Intact plant or bacterial toxins linked to antibodies may acquire some cell selectivity but because toxin binding sites are retained, non-specific toxicity can still occur. Since the binding region of such toxins is distinct from the enzymatically active toxic portion, these components can be cleaved and isolated (Olsnes and Pihl, 1973). Thus, purified toxic subunits can be coupled to antibodies to produce highly selective cytotoxic agents which are devoid of residual toxin binding.

Intracellular Pathways of Ricin A Chain Cytotoxins

Ricin A chain, a potent ribosomal poison, was disulfide linked either to the iron transport protein, transferrin, or to anti-transferrin receptor antibodies to produce highly specific derivative toxins, Tf-A and TfR-A, respectively. The ability of these agents to gain access to and damage ribosomes within the cell was accelerated in the presence of carboxylic ionophores. Their effectiveness for killing clonogenic target cells was correspondingly enhanced by 5 logs after a brief treatment with Tf-A plus ionophore. Intracellular trafficking of Tf-A and TfR-A was monitored by a variety of methods to better understand their mechanism of action. Data obtained with 125I-labeled A chain and 59Fe3+-labeled toxin probes indicated that the natural iron delivery pathway was initially followed. This was characterized by specific attachment to surface receptors, internalization, entry into low-density acidic vesicles, uncoupling of iron, an absence of lysosomal degradation, and sustained cycling. Ultrastructural studies using a colloidal gold-labeled anti-A chain probe confirmed the presence of these toxins within the structural elements associated with endocytosis. Toxic Tf-A molecules, however, diverged from this pathway (t1/2 = 88 min) to eventually kill cells as witnessed by a gradual loss in the ability to rescue cells using excess transferrin. Potentiating agents, such as carboxylic ionophores or B chain, seem to act by speeding the divergence of Tf-A and TfR-A from the normal endocytotoxic cycle.

Study of the Transferrin Receptor Using a Cytotoxic Human Transferrin-Ricin a Chain Conjugate

The A chain of ricin which catalytically inactivates eukaryotic ribosomes inhibits cellular protein synthesis only after entering the cell. Although attachment of pure A chain to cell membranes has not been demonstrated (Raso, 1981), it is toxic at high concentrations indicating that cells can inefficiently internalize this 30,000 MW protein. Cytotoxicity is enhanced 103–105-fold however by recombining A chain either with its natural B chain subunit or with alternative carriers which deliver it to the cell surface and facilitate access to ribosomes within the cytoplasm. Thus, cyto-toxins possessing different cell receptor selectivities can be designed by coupling A chain to antibody or ligand carriers with appropriately chosen specificity characteristics. This controlled cytotoxic action has stimulated interest from the standpoint of eliminating undesirable cells for therapeutic reasons but is equally important for cell biology studies.