Cytotoxic Potency of CD22‐Ricin A Depends on Intracellular Routing Rather than on the Number of Internalized Molecules

Abstract

van Horssen PJ, van Oosterhout YJM, de Witte T, Preijers FWMB. Cytotoxic Potency of CD22-Ricin A Depends on Intracellular Routing Rather than on the Number of Internalized Molecules. Scand J Immunol 1995;41:559-65 Cytotoxicity of immunotoxins (ITs) varies considerably depending on factors like the capability of the target antigen to internalize IT molecules, intracellular processing and routing of the IT. We studied factors that may influence cytotoxicity of CD22-ricin A IT to several B cell lines. The antigen density varied from 5.9xlO3 to 6.0xlO4 molecules/cell. The ID 50, determined by protein synthesis inhibition, varied from 2.1xl0-12 to 3 .8 x l0 " n M IT in absence and from 2 .8 x l0 -14 M to 5 .2 x l0 " 12 M IT in presence of the cytotoxicity enhancer N H 4CI (6 mM). In absence as well as in presence of N H 4CI no correlation could be found between antigen density and ID 50. No relation was observed either with the rate of cytotoxicity. Even in cell lines with a low antigen density, such as KM3, protein synthesis was quickly inhibited. In order to investigate whether the cytotoxicity was dependent on the number of internalized molecules the kinetics of internalization and exocytosis of degraded 125I-labelled CD22 molecules were studied. After 24h the number of internalized CD 22 molecules was highest in Ramos (154,500), followed by Daudi (110,300) and KM3 (69,900). However, despite the higher internalization rate of Daudi the rate of cytotoxicity of 10" s M IT was comparable with KM3. N H 4CI did not influence the number of internalized molecules but postponed degradation of CD22. In conclusion, CD22-ricin A is a very potent and fast acting IT even for elimination of target cells that express low numbers of antigen. These results may have implication for treatment of different B cell malignancies with CD22-ricin A.

True

“…Similar contradictory results have been obtained with NH 4 Cl. 6 Monensin, nigericin, NH 4 Cl, chloroquine and bacitracin all enhanced the activity of CD22-rec ricin A. Nigericin was more potent than the combination of chloroquine and NH 4 Cl, which in turn was more potent than monensin and bacitracin. The combination of chloroquine and NH 4 Cl was more potent than both agents individually.…”

“…5 Upon binding, the Bl-CAM/IT complex is rapidly internalized, whereafter the antigen is re-expressed. 6,7 This makes Bl-CAM an optimal target for ITs. Several investigators have shown that CD22 ITs have a strong anti-B cell potency in vitro as well as in vivo.…”

“…Several investigators have shown that CD22 ITs have a strong anti-B cell potency in vitro as well as in vivo. 6,[8][9][10][11][12][13][14] These findings resulted in clinical phase I/II studies. [15][16][17][18] Though achieving some complete responses, treatment of these patients did not result in complete cure.…”

Influence of cytotoxicity enhancers in combination with human serum on the activity of CD22-recombinant ricin A against B cell lines, chronic and acute lymphocytic leukemia cells

“…30,40 Inhibition of activity might also be induced by covalent binding of ␣ 2 -macroglobulin to rec ricin A as has been described for deglycosylated ricin A chain and its ITs by Ghetie et al 45 who found a substantial amount of complexes formed after 24 h. However, we could not detect these high molecular complexes with the immunoblotting assay used. Besides, the kinetics of CD22-rec ricin A activity are too fast 6 to allow these complexes to form. 45 This suggests that more factors contribute to the reduction of IT activity by serum.…”

“…Previously we showed that 6 mm NH 4 Cl enhanced the CD22-rec ricin A activity to eight different B cell lines between seven-and 93-fold. 6 Chloroquine enhanced the activity of CD22-rec ricin A in a dose-dependent fashion (Table 3) with 50 m chloroquine enhancing the IT activity 14-fold for Daudi cells and 11-fold for Ramos cells.…”