title: Cytotoxic potency of CD22-ricin A depends on intracellular routing rather than on the number of internalized molecules.
abstract: 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 5.9 x 10(3) to 6.0 x 10(4) molecules/cell. The ID50, determined by protein synthesis inhibition, varied from 2.1 x 10(-12) to 3.8 x 10(-11) M IT in absence and from 2.8 x 10(-14) M to 5.2 x 10(-12) M IT in presence of the cytotoxicity enhancer NH4Cl (6 mM). In absence as well as in presence of NH4Cl no correlation could be found between antigen density and ID50. 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 24 h the number of internalized CD22 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(-8) M IT was comparable with KM3. NH4Cl 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.
authors.affiliation: Department of Hematology, University Hospital St Radbound, Nijmegen, The Netherlands.
authors.forename: P J
authors.initials: PJ
authors.lastname: van Horssen
authors.forename: Y V
authors.initials: YV
authors.lastname: van Oosterhout
authors.forename: T
authors.initials: T
authors.lastname: de Witte
authors.forename: F W
authors.initials: FW
authors.lastname: Preijers
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000378:metabolism / Q000633:toxicity; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism / Q000633:toxicity; D001402:B-Lymphocytes / Q000276:immunology; D015815:Cell Adhesion Molecules; D002460:Cell Line; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D006801:Humans; D000922:Immunotoxins / Q000378:metabolism* / Q000633:toxicity*; D037102:Lectins; D012276:Ricin / Q000378:metabolism* / Q000633:toxicity*; D051918:Sialic Acid Binding Ig-like Lectin 2
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; D015815:Cell Adhesion Molecules; D000922:Immunotoxins; D037102:Lectins; D051918:Sialic Acid Binding Ig-like Lectin 2; D000643:Ammonium Chloride; D012276:Ricin