title: Conjugation of blocked ricin to an anti-CD19 monoclonal antibody increases antibody-induced cell calcium mobilization and CD19 internalization.
abstract: CD19 (B4) is a signal transduction molecule restricted to the B-cell lineage and the target of the immunotoxin anti-B4-blocked ricin (anti-B4-bR), which is composed of the monoclonal antibody (MoAb) anti-B4 and the modified plant toxin blocked ricin. To explore the influence of conjugation of blocked ricin to anti-B4 on functional activation of CD19, we investigated the effects of anti-B4-bR, and that of unconjugated anti-B4, on intracellular calcium mobilization and ligand/receptor internalization. The data showed that anti-B4-bR was more potent than anti-B4 in triggering cell calcium mobilization. Two other immunotoxins that bind to the B-cell surface, anti-CD20-bR and anti-CD38-bR, were devoid of the calcium increasing effect of anti-B4-bR. Furthermore, anti-B4 conjugated to ricin A-chain was also without effect in Namalwa cells, indicating that the ricin B-chain component was required for anti-B4-bR effect. Anti-B4-bR-induced calcium mobilization was inhibited in the presence of lactose, yet the calcium response induced by cross-linking anti-B4-bR with a second step antibody was not affected. The extent of CD19 modulation induced by anti-B4-bR was higher than that induced by anti-B4, and lactose dampened the effect of the immunotoxin down to that of the MoAb. Moreover, the number of internalized immunotoxin molecules was higher than that of unconjugated MoAb. Although a mechanism involving dimerization of the immunotoxin cannot be excluded, our findings suggest that the residual binding activity of the blocked ricin B-chain to cell surface molecules plays an important role in the greater calcium fluxes and greater internalization rate of anti-B4-bR, and is of functional significance in the mechanism of intoxication of cells by the immunotoxin.
authors.affiliation: Maisonneuve-Rosemont Hospital Research Center, Montreal, Quebec, Canada.
authors.forename: A C
authors.initials: AC
authors.lastname: Goulet
authors.forename: V S
authors.initials: VS
authors.lastname: Goldmacher
authors.forename: J M
authors.initials: JM
authors.lastname: Lambert
authors.forename: C
authors.initials: C
authors.lastname: Baron
authors.forename: D C
authors.initials: DC
authors.lastname: Roy
authors.forename: E
authors.initials: E
authors.lastname: Kouassi
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015703:Antigens, CD; D018941:Antigens, CD19 / Q000502:physiology*; D018951:Antigens, CD20 / Q000276:immunology; D000943:Antigens, Differentiation / Q000276:immunology; D002118:Calcium / Q000378:metabolism*; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry* / Q000378:metabolism; D007785:Lactose / Q000494:pharmacology; D008562:Membrane Glycoproteins; D009244:NAD+ Nucleosidase / Q000276:immunology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012276:Ricin / Q000737:chemistry*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018941:Antigens, CD19; D018951:Antigens, CD20; D000943:Antigens, Differentiation; D004791:Enzyme Inhibitors; D000922:Immunotoxins; D008562:Membrane Glycoproteins; D011500:Protein Synthesis Inhibitors; D012276:Ricin; D011505:Protein-Tyrosine Kinases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1; D007785:Lactose; D002118:Calcium