Drug-Dependent Murine Monoclonal Antibodies (mAbs) Mimic Antibodies Found in Patients with Drug-Induced Immune Thrombocytopenia (TP).

Abstract

Drug-induced immune thrombocytopenia (DITP) is a serious and sometimes life-threatening complication of treatment with many drugs. In most instances (excluding heparin-induced TP), platelet (plt) destruction is caused by antibodies (abs) that recognize distinct epitopes on platelet membrane glycoproteins (GP) only when the sensitizing drug is present in soluble form. How drug at pharmacological levels promotes tight binding of antibody to a specific target is unknown, in part because only polyclonal human abs have been available for study. We sought to produce monoclonal abs (mAbs) that mimic the behavior of human drug-dependent abs to create tools that can be used to study the molecular basis for this interaction. Mice were immunized with GPIIb/IIIa isolated from human platelets together with soluble quinine (Qn), tirofiban (Tf), or eptifibatide (Ef), three drugs that commonly cause DITP. Hybridomas were prepared from splenic B cells using a standard protocol and approximately 550 supernatants from each cultured hybrid line were screened for DDAbs by flow cytometry using normal platelets as targets. To date, 11 Abs that react with GPIIb/IIIa only in the presence of the immunizing drug (2 Qn-, 3 Tf- and 6 Ef-specific) have been identified. Preliminary studies show that the Qn-specific abs bind reversibly to GPIIb/IIIa at 50 nM Qn, a concentration much lower than is achieved pharmacologically, and are not inhibited by Qn at 5 mM, the limit of solubility. Quinidine (Qd) the diastereoisomer of Qn, supports only weak ab binding at a concentration of 50 uM. The tirofiban and eptifibatide-dependent abs recognize GPIIb/IIIa occupied by these RGD ligand-mimetic GPIIb/IIIa inhibitors. In each of these respects, reaction patterns of the three groups of mAbs closely resemble those of abs from patients experiencing TP after treatment with one of these drugs. These findings show that mAbs mimicking the behavior of human drug-dependent abs can be produced by immunizing mice with GP and soluble drug to produce probes suitable for characterizing the molecular basis of ab-drug-target interactions leading to platelet destruction in DITP. It is noteworthy that these mAbs were induced using soluble drug and protein for immunization because this suggests that the immune response leading to DITP does not require the sensitizing drug to be covalently linked to a protein, i.e, does not require the drug to act as a classical hapten.