CYTOTOXIC POTENCY OF CD22-RICIN-A DEPENDS ON INTRACELLULAR ROUTING RATHER THAN ON THE NUMBER OF INTERNALIZED MOLECULES

Cytotoxicity of immunotoxins (ITs) varies considerably depending on fa ctors like the capability of the target antigen to internalize IT mole cules, intracellular processing and routing of the IT. We studied fact ors that may influence cytotoxicity of CD22-ricin A IT to several B ce ll lines. The antigen density varied from 5.9x10(3) to 6.0x10(4) molec ules/cell. The ID50, determined by protein synthesis inhibition, varie d from 2.1x10(-12) to 3.8x10(-11) M IT in absence and from 2.8x10(-14) M to 5.2x10(-12) M IT in presence of the cytotoxicity enhancer NH4Cl (6 mM). In absence as well as in presence of NH4Cl no correlation coul d be found between antigen density and ID50. No relation was observed either with the rate of cytotoxicity. Even in cell lines with a low an tigen density, such as KM3, protein synthesis was quickly inhibited. I n order to investigate whether the cytotoxicity was dependent on the n umber of internalized molecules the kinetics of internalization and ex ocytosis of degraded I-125-labelled CD22 molecules were studied. After 24 h the number of internalized CD22 molecules was highest in Ramos ( 154,500), followed by Daudi (110,300) and KM3 (69,900). However, despi te the higher internalization rate of Daudi the rate of cytotoxicity o f 10(-8) M IT was comparable with KM3. NH4Cl did not influence the num ber of internalized molecules but postponed degradation of CD22. In co nclusion, CD22-ricin A is a very potent and fast acting IT even for el imination of target cells that express low numbers of antigen. These r esults may have implication for treatment of different B cell malignan cies with CD22-ricin A.