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.