Internalisation and trafficking characteristics of CD7 and CD38 on a human T-ALL cell line in relation to immunotoxin potency

Abstract

In this study, the enhanced cytotoxic effect of the anti-CD7 IT HB2-Saporin and the anti-CD38 IT OKT10-Saporin used in combination when compared to their use individually was demonstrated on the human T-ALL cell line HSB-2 both in vivo and in vitro. In an in vitro cell proliferation assay and an in vivo SCID mouse model, OKT10-Saporin was shown to be more effective than HB2-Saporin, but neither individual IT was as potent as their combined use. In contrast, HB2-Saporin performed best in the short term protein synthesis assay (PSI) and the combination of two ITs demonstrated an intermediate potency between that of the two individual ITs. Three explanations were proposed for the improved efficacy of using two ITs simultaneously: 1) co-ligation of CD7 and CD38 might alter the individual internationalisation characteristics; 2) targeting against two molecules overcomes the heterogeneity of antigen expression on tumour cells or 3) using two immunotoxins increases the amount of toxin delivered to the target cell. It is possible that all three explanations are valid.

Flow cytometry and confocal microscopy were used to determine the internalisation and intracellular routing characteristics of CD7 and CD38 on the T-ALL cell line HSB-2, when ligated by antibody individually or in combination. These studies indicate that CD7 and CD38 have very different internalisation kinetics. CD7 clears very rapidly from the cell surface following ligation by antibody, whereas only 50% of CD38 molecules internalise over a 24 hr duration. When CD7 and CD38 were ligated by antibody simultaneously, no change to the internalisation characteristics or intracellular routing of either antibody appeared altered. In these studies it has become clear that there is no direct correlation between internationalisation, intracellular routing and cytotoxic potency.

Brefeldin A was used as a tool to investigate what intracellular routes these ITs may follow within the cell. These were preliminary studies and further work is required.

These studies have revealed that no single factor determines IT potency. Internalisation rate is only important if degradation is avoided and trafficking occurs to an appropriate intracellular compartment from which the toxin component can translocate.

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