Triple tandem repeats of the sequence LESIINFEKLTDFSGSSCSSCRDQRGWP, which includes the MHC class I epitope (OVA-I), are important for antigen presentation in vitro.

(A) Schematic drawing of nine artificial proteins used in the second screening; the amino acid sequences of these proteins are shown in Figure S3, Supporting Information. Underlines show the common structure (triple tandem repeats of the class I epitope and peptide sequence produced by the cybergene program) present in F182A, F37A and F36C. (B) OVA-specific T-cell activation in vitro by F37A and F36C. (C) Schematic drawings of sequences present in F37A and its mutants. The OVA-I sequences (SIINFEKL) (red) of F37A were replaced with one or more WT1 epitopes (RMFNAPYL) (yellow), one-by-one. (D) Determination of OVA-specific antigenicity of the mutant proteins using in vitro antigen presentation assay. When DC2.4 cells were treated with different concentrations (10 µg/ml, 20 µg/ml and 40 µg/ml), of the indicated antigens, only F37A and F37AE2, but not the other mutants, induced IL-2 production. Only the five C-terminal amino acids of F37AE2 differed from F37A.