title: A bioassay for the detection of neutralizing antibodies against the α-melanocyte stimulating hormone analog afamelanotide in patients with erythropoietic protoporphyria.
abstract: The tridecapeptide afamelanotide (Scenesse®) is a congener of α-melanocyte stimulating hormone (α-MSH). Upon binding to the melanocortin 1 receptor (MC1R) on the surface of pigment cells of the skin, the melanocytes, α-MSH or afamelanotide trigger the synthesis of cAMP, which stimulates the synthesis of melanin and therefore induces skin tanning. In a recent trial, afamelanotide administered as controlled release implants protected erythropoietic protoporphyria (EPP) patients from sunlight induced phototoxic skin reactions. Administration of biological therapeutic peptides may elicit unwanted immunogenic responses in recipients of these products. Although in a previous study using ELISA technique we excluded any newly developed immunogenicity during prolonged exposure to afamelanotide, we confirmed the previously published existence of low titers of antibodies against α-MSH in drug-naïve individuals that cross-reacted with afamelanotide. In order to investigate whether such antibodies are neutralizing, i.e. could block the biological effect of afamelanotide, we developed a cell culture-based bioassay. The basis of our assay was the measurement of afamelanotide-induced cAMP formation in a strain of the B16 mouse melanoma cell line, G4F-7, expressing the transfected human MC1R. Average half-effective concentrations of the natural hormone α-MSH and its congener afamelanotide were 38.8 ± 10.6 and 10.9 ± 7.17 nM (n=5), respectively. Neutralizing antibodies would reduce the cAMP formation. Two neutralizing anti-α-MSH antibodies served as positive controls. cAMP formation in the G4F-7 cells after addition of sera of drug-naïve (n=6) and of drug-exposed EPP patients (n=17) was significantly lower than after that from healthy volunteers (n=13). There was no difference between drug-naïve and drug-exposed patients. Using forskolin as a hormone-independent stimulator of cAMP formation, we excluded an unspecific interference of EPP sera with cAMP formation. We conclude that afamelanotide even after prolonged application to EPP patients did not elicit neutralizing antibodies. Further, the low titer immunoreactivity observed in sera of some drug-naïve individuals had no effect on the biological activity of afamelanotide.
authors.affiliation: Institute for Laboratory Medicine, Stadtspital Triemli, Zurich CH-8063, Switzerland.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Spichty
authors.forename: Mirjam
authors.initials: M
authors.lastname: Balimann
authors.forename: Jasmin
authors.initials: J
authors.lastname: Barman
authors.forename: Elisabeth I
authors.initials: EI
authors.lastname: Minder
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000032:analysis*; D045744:Cell Line, Tumor; D003429:Cross Reactions; D000242:Cyclic AMP / Q000378:metabolism; D003879:Dermatologic Agents / Q000037:antagonists & inhibitors* / Q000494:pharmacology / Q000627:therapeutic use; D006801:Humans; D017496:Hypopigmentation / Q000209:etiology / Q000517:prevention & control; D008544:Melanocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D015166:Monitoring, Immunologic; D009994:Osmolar Concentration; D017092:Porphyria, Erythropoietic / Q000097:blood / Q000188:drug therapy / Q000276:immunology* / Q000503:physiopathology; D044102:Receptor, Melanocortin, Type 1 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D057134:Antibodies, Neutralizing; D003879:Dermatologic Agents; D044102:Receptor, Melanocortin, Type 1; D011994:Recombinant Proteins; D000521:alpha-MSH; D000242:Cyclic AMP; C534526:afamelanotide