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Assesses the immune response to peptide vaccination among melanoma patients in Bethesda, Maryland. Use of real time quantitative polymerase chain reaction assay; Relevance of detecting tumor antigen response in developing cancer vaccines; Implication of the assessment in understanding tumor-host interaction.

Immune responses against tumor-associated antigens rely on efficient epitope presentation. The melanoma-associated antigen (Ag) gp100 contains HLA-A*0201 ligands that are characterized by low to medium binding affinity, among which gp100209-217 is the most prominent (Kawakami et al., J Immunol 154:3961–3968, 1995). While this epitope is a natural T-cell target, it primes with low-efficiency T-cell responses during immunization. A modified gp100 epitope, gp100209-217T210M, that contains a Thr to Met substitution at position 2 of the antigenic nonamer is characterized by high binding affinity for HLA-A*0201 and elicits strong and clinically effective T-cell responses. This higher affinity is believed to represent the sole reason for enhanced immunogenicity. Contrasting with this observation is the unpredictable relationship between affinity and immunogenicity observed in other antigen systems. In addition, we noted a striking difference between the capability of endogenously processed gp100209-217 and gp100209-217T210M to induce T-cell responses in an in vitro model. Therefore, we questioned whether factors other than HLA-affinity might play a role in determining the immunogenicity of these epitopes. In the present study, we evaluated the in vitro proteasomal cleavages of 23meric precursor peptides encompassing the native sequence (gp100201-223) or the modified sequence (gp100201-223T210M). Here we show that the standard proteasome liberates the C-termini of both antigenic peptides but not the N-termini. Quantitative analysis of the digestion products revealed that more of the fragments displaying the final C-termini were produced from the wild-type precursor. However, a stronger TCR engagement was observed when fractions of digested gp100201-223T210M were used to activate an HLA-A*0201–expressing target T-cell clone. This difference was also found using separately produced, synthetic nonamers. In conclusion, the high binding affinity of gp100209-217T210M seems to compensate for possible differences in proteasomal cleavage at the biological level. Since the final antigenic nonamer is not directly produced by the proteasome, additional further factors may influence the antigenic peptide availability, such as post-proteasomal processing and intracellular peptide transport. [ABSTRACT FROM AUTHOR]