FITC
Excitation: 490nm, Emission: 525nm
Cetuximab is remarkable for the relatively high rate and severity of hypersensitivity reactions (HR) being reported in the literature. Screening for cetuximab-specific IgE in serum via immunoassay has been found to be useful in preventing HR; however, these tests are known to have a low positive predictive rate. In an attempt to remedy this, we evaluated the interaction between cetuximab and IgE on basophils for predicting severe cetuximab-induced HR. Twelve head and neck cancer patients were enrolled in this single-institution study: four with a history of cetuximab-induced HR and eight with no such history. Cetuximab-specific and galactose-α-1,3-galactose (α-gal) specific IgEs in serum were measured in vitro using an enzyme-linked immunosorbent assay (ELISA). IgE-cetuximab binding on basophils was also analyzed to evaluate the decrease in cetuximab molecules on basophils after dissociation of IgE from FcεRI. The positive predictive value associated with the presence of cetuximab- or α-gal-specific IgE in serum was found to be only 0.67, whereas the negative predictive value was 1.00. On the other hand, in all four patients who developed HR, the cetuximab molecules on basophils were decreased significantly due to the dissociation of IgE from basophils (P < 0.05). However, this was not the case in patients who did not develop HR. In conclusion, our results strongly imply that the IgE-cetuximab interaction on basophils may be key to developing improved methods for predicting severe cetuximab-induced HR.
© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
Immunoglobulin E-mediated allergy and certain autoimmune diseases are characterized by the presence of a T helper type 2 (Th2) immune response and allergen-specific or self-reactive IgE. Soluble CD23 (sCD23) is a B-cell factor that fosters IgE class-switching and synthesis, suggesting that sCD23 may be a therapeutic target for these pathologies. We produced a recombinant protein, CTLA4Fcε, by fusing the ectodomain of the immunoregulatory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) with a fragment of the IgE H-chain constant region. In SDS-PAGE/inmunoblot analyses, CTLA4Fcε appeared as a 70,000 MW polypeptide that forms homodimers. Flow cytometry showed that CTLA4Fcε binds to IgE receptors FcεRI and FcεRII/CD23, as well as to CTLA-4 counter-receptors CD80 and CD86. Binding of CTLA4Fcε to FcεRII/CD23 appeared stronger than that of IgE. Since the cells used to study CD23 binding express CD80 and CD86, simultaneous binding of CTLA4Fcε to CD23 and CD80/CD86 seems to occur and would explain this difference. As measured by a human CD23-specific ELISA, CTLA4Fcε - but not IgE - induced a concentration-dependent reduction of sCD23 in culture supernatants of RPMI-8866 cells. Our results suggest that the simultaneous binding of CTLA4Fcɛ to CD23-CD80/CD86 may cause the formation of multi-molecular complexes that are either internalized or pose a steric hindrance to enzymatic proteolysis, so blocking sCD23 generation. CTLA4Fcε caused a concentration-dependent reduction of lymphocyte proliferation in human peripheral blood mononuclear cell samples stimulated in vitro with concanavalin A. The ability to bind IgE receptors on effector cells, to regulate the production of sCD23 and to inhibit lymphocyte proliferation suggests that CTLA4Fcɛ has immunomodulatory properties on human Th2 responses.
© 2016 John Wiley & Sons Ltd.