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Cytotoxicity of CD3-Ricin A Chain Immunotoxins in Relation to Cellular Uptake and Degradation Kinetics

Department of Hematology, University Hospital St. Radboud, Geert Grooteplein 8, 6500 HB Nijmegen, the Netherlands

The cytotoxicity of WT32 (CD3)-ricin A immunotoxin (IT) to the acute lymphoblastic leukemia T-cell line Jurkat was compared with the rate of internalization and intracellular degradation of WT32 and WT32-ricin A during continuous exposure. Moreover, the influence of NH₄Cl and monensin on these processes was studied.

Based on protein synthesis inhibition ([³H]leucine incorporation), it appeared that cytotoxicity was not fully expressed directly after exposure to IT due to a delay in either the internalization of membrane-bound IT or the action of intracellular ricin A. Varying the duration of incubation and postponing [³H]leucine addition for up to 24 h after initiation showed that cytotoxicity occurred in two phases, rapid internalization of initially bound IT followed by a continuous but slower uptake, possibly due to reexpression of the CD3 antigen.

No differences were found in the rate of internalization and degradation of ¹²⁵I-labeled WT32 and WT32-ricin A. Internalization started rapidly after binding at 37°C, was fastest during the first 12 h (±360,000 molecules/cell), and continued for at least 24 h (±420,000 molecules/cell). Exocytosis of intracellularly degraded molecules became measur-able after 1 to 2 h of incubation at 37°C and increased to approximately 400,000 molecules/cell in 24 h. After 4 h of incubation at 37°C the number of internalized molecules exceeded the amount of WT32 that could maximally bind to the cell membrane (±150,000 molecules/cell), confirming reexpression of antigen.

The addition of NH₄Cl and monensin enhanced the cytotoxicity of WT32-ricin A, probably due to an increased intracellular amount of IT. These agents appeared to reduce strongly the degradation of internalized WT32, resulting in an accumulation of intracellular molecules. NH₄Cl was most effective during the first 12 h of incubation, whereas monensin increased the amount of intracellular WT32 molecules after 2 to 24 h.

Our observations suggest that incubation conditions for the optimal cytotoxicity of IT treatment can be predicted by studying the internalization and degradation of the IT or respective monoclonal antibody.

¹ To whom requests for reprints should be addressed.

Received 3/11/92. Accepted 8/25/92.

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