Stimulation of tumor-reactive T lymphocytes using mixtures of synthetic peptides derived from tumor-associated antigens with diverse MHC binding affinities.

The use of reverse immunology may be necessary to identify new tumor-associated antigens, particularly for cancers, against which tumor-reactive T cell populations have been difficult to establish. One approach has been to screen peptides derived from a candidate antigen with high major histocompatibility complex (MHC) binding affinities for the induction of tumor-reactive T lymphocytes in vitro. However, many candidate antigens that are overexpressed in tumors are nonmutated self-proteins, and unlike foreign or mutated proteins, immunodominant epitopes may not be expressed at high density on the surface of tumor cells. Therefore, to identify tumor-associated epitopes, it may be necessary to screen large panels of peptides with wide ranges of MHC binding affinities. The current methodology of stimulating peripheral blood lymphocytes (PBL) from donors expressing the MHC molecule of interest with individual peptides is impractical for screening such large panels. Therefore, we evaluated the use of mixtures of peptides with variable MHC binding affinities for the induction of tumor-reactive T lymphocytes with the melanoma antigens gp100 and an alternate isoform of tyrosinase-related protein 2 (TRP2-6b) as models. A mixture of 10 known human leukocyte antigen (HLA)-A*0201-restricted peptides from gp100 induced melanoma-reactive cytotoxic T lymphoycte (CTL) from multiple patients with metastatic melanoma. The majority of these T cell populations recognized the known immunodominant epitopes gp100:209-217 and gp100:280-288, even though the HLA-A*0201 binding affinities of these peptides were much lower than other peptides in the mixture. Similarly, melanoma-reactive CTL were generated with a mixture of HLA-A*0201-restricted peptides from TRP2-6b, and these responses were directed against the previously identified tumor-associated epitopes TRP2-6b:180-188, TRP2-6b:288-296 and TRP2-6b:403-411. These results suggest that the use of peptide mixtures may facilitate the identification of new tumor-associated antigens through the application of reverse immunology.