The likelihood of identifying peptides of sufficient quality for the development 
of effective cancer vaccines by screening of phage display libraries is low. 
Here, we introduce the sequential application of systematic amino acid 
substitution by SPOT synthesis. After the substitution of two amino acids within 
the sequence of a phage display-derived mimotope of disialoganglioside GD2 
(mimotope MA), the novel mimotope C3 showed improved GD2 mimicry in vitro. 
Peptide vaccination with the C3 mimotope induced an 18-fold increased anti-GD2 
serum response associated with reduction of primary tumour growth and 
spontaneous metastasis in contrast to MA mimotope controls in a syngeneic 
neuroblastoma model. In summary, SPOT provides an ideal optimisation tool for 
the development of phage display-derived cancer vaccines.