Cancers in humans are commonly associated with mutations in dominant and recessive oncogenes. These genes produce mutated proteins that are unique to cancer cells. Ras proto-oncogenes are the best characterized mutated genes in human cancer (22-26). They encode a highly conserved family of 21 Kd proteins (p21). With a single amino acid mutation, the ras protein can potentiate transforming capabilities both in mouse and human cells. Such point mutated ras have been found in a broad spectrum of human carcinomas notably at codons 12, 13 and 61. Codon 12 mutations form more than 90% of all ras mutations in human cancers.
Point mutations in the ras p21 proto-oncogenes (i.e., K-ras, H-ras, N-ras) have been identified, described and associated with a high frequency and spectrum of human cancers, including adenocarcinomas of the pancreas, colon and lung as well as melanomas and myeloid leukemias (reviewed in References 1-4). Such mutations lead to the production of aberrant proteins, which are distinct from normal endogenous ras p21 in both structure (DNA and protein sequences) and function and represent early events in cellular transformation. In human carcinomas which harbor p21 point mutations, it is the K-ras gene at codon 12 that is found frequently mutated, whereby the normal glycine (Gly) residue is replaced with either an aspartic acid (Asp), valine (Val), cysteine (Cys), alanine (Ala), arginine (Arg) or serine (Ser) residue (1,2,4). Substitutions of Gly to Asp, Val and Cys, collectively, however, account for the majority of human carcinomas with such p21 position 12 point mutations (1,2,4).
T Lymphocytes can recognize antigens (Ag) presented in the context of major histocompatibility complex (MHC) class I or class II molecules on the surface of antigen presenting cells (APC) (27-31). These surface Ag are thought to be short peptides that are derived from degraded intact proteins (32-34).
T lymphocytes are divided into two major populations, CD4+ and CD8+. The CD8+ T cells recognize peptides (8-10 residues) bound to MHC class I molecules and are associated with cytotoxic activity (30-31). The CD4+ lymphocytes are generally involved in the recognition of peptides (13-18 residues) presented in the context of MHC class II molecules (28,29), and are involved in immunoregulation through cytokine secretion. Th1, a subset of CD4+ T cells have been reported to express lytic activity (35-37).
Data in mice has shown the generation of MHC restricted (specific) cytotoxic T cells that are capable of detecting endogenous, cytoplasmic peptide antigens (antigens presented from within the cell membrane on the cell surface)38,39. These T cells can cause rejection (lysis) of cells expressing such peptides. This rejection is mediated by cells responding to novel peptides derived from mutated genes (38,39). T cells are capable of detecting single amino acid discrepancies between homologous peptides presented on APC (40,41).
The generation and expression of these previously unseen, “neo-determinants” may now represent unique and highly specific epitopes for T cell (CD4+ and/or CD8+) recognition, which has been proposed in host defense as an important effector pathway in the control of malignancy (5). Studies in both murine and human systems, have previously identified and characterized immunodominant CD4+ T cell epitopes of mutant K-ras at codon 12 using short synthetic peptides (6-12). With respect to human anti-ras CD8+ cytotoxic T lymphocyte (CTL) responses, however, nothing is yet defined for position 12 mutations, although one has been reported for position 13 and one for position 61 (13, 14). Thus, the identification herein of human CD8+ T cell epitopes reflecting specific ras p21 point mutations has important and direct implications for the development of oncogene-specific vaccines in cancer immunotherapy.