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Blocking Effect of Human Serum but not of Cerebrospinal Fluid on Ricin A Chain Immunotoxin Potentiation by Monensin or Carrier Protein-Monensin Conjugates¹

Istituto di Scienze Immunologiche [C. C., A. F., R. C., M. P., C. A., G. T., M. C.] and Istituto di Farmacologia [G. B.], University of Verona, Policlinico Borgo Roma, 37134 Verona, Italy

The potentiation of monoclonal antibody/ligand toxin (immunotoxin) cytotoxicity by the ionophore monensin (Mo) or by human serum albumin-monensin (HSA-Mo) conjugates was investigated. Since disulfide cross-linked HSA-Mo (HSA-SPDP-Mo) is rapidly inactivated by human serum (M. Colombatti et al., Cancer Res., 50: 1385–1391, 1990), we synthesized thioether cross-linked HSA-Mo conjugates (HSA-SIA-Mo). HSA-SIA-Mo is resistant to treatment with reducing agents (e.g., glutathione, dithiothreitol) and shows potentiating activity identical to that of Mo or of HSA-SPDP-Mo, enhancing immunotoxin (IT) cytotoxicity 45-35,000-fold.

Human leukemic and tumor cell lines are highly sensitive to treatment with IT in combination with Mo, HSA-SPDP-Mo, or HSA-SIA-Mo (concentration required to inhibit protein synthesis by 50%, 10^–10-2.5 x 10^–13 M).

IT potentiation by both types of HSA-Mo conjugates, however, is inhibited by whole human serum. In contrast, human cerebrospinal fluid has no effect on the potentiation of IT by Mo or HSA-Mo conjugates. The serum blocking factors reside mostly in a M_r 40,000–90,000 protein fraction. Serum components of low molecular weight (<10,000) show no detectable effect upon the stability of HSA-Mo conjugates.

The toxicity of HSA-SIA-Mo in vivo was investigated by intrathecal injections in rats. Concentrations of up to 60 µg/kg can be injected into the brain with only transient neurological sequelae.

We therefore conclude that if the systemic delivery of HSA-Mo conjugates for the potentiation of ricin A chain-IT presents some limitations due to the blocking effect of serum, the application of HSA-Mo conjugates in combination with ricin A chain-IT for regional tumor therapy in the brain appears more promising.

¹ This work was supported in part by grants from CNR (PF Ingegneria Genetica, PF Biotecnologie e Biostrumentazione), Associazione Italiana per la Ricerca sul Cancro, M.P.I. 40% Aspetti Clinico-Sperimentali della Risposta Immune, M.S. Progetto AIDS, and Associazione per la promozione delle Ricerche Biomediche.

² R. C. is a recipient of an AIRC Fellowship.

³ To whom requests for reprints should be addressed.

Received 7/15/91. Accepted 11/ 5/91.

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