A high‐throughput genetic system for identifying enhanced altered peptide ligands of tumor associated antigens: implications for enhancing adoptive immunotherapy

Abstract

Cancer immunotherapy offers a promising approach for eradicating metastatic tumors that fail to fully respond to existing therapies. Unfortunately, efforts are frequently hampered by tolerance mechanisms that act to prevent autoimmunity. A potential means for circumventing these mechanisms is the use of altered peptide ligands (APL). APLs are mutant T cell epitopes that can either enhance or dampen a T cell response. Existing methods for identifying effective APLs are either limited in scope or are expensive and technically restrictive. The objective of this study is to develop a comprehensive genetic approach for identifying enhanced APLs of CTL epitopes. With this procedure every point mutant of a CTL epitope can be generated and screened, and the effective APL easily identified. In this study we utilize saturation mutagenesis to independently mutagenize every codon of Mart‐126–35 A27L. Recombinant mini‐gene products of the APLs are cross‐presented to Mart‐126–35 ‐specific CTL clones via dendritic cells in a 96‐well based assay format. Here we identify three novel superagonist peptides of Mart‐126–35, which can generate higher frequencies of Mart‐126–35 ‐specific CTL from peripheral blood leukocytes of melanoma patients. This work has implications for the identification of APLs that may prove clinically beneficial in generating tumor‐specific CTLs for use in adoptive T cell immunotherapy.