Cancer antigens containing mutations in an expressed gene of cancer cells from a cancer patient are identified. Sequences from cancer cells obtained using a parallel sequencing platform are selected by comparing to the patient's normal genes or to normal genes from an HLA-matched individual. Sequences are further selected by identifying an HLA supertype of the cancer patient and selecting for that HLA supertype, sequences that have a particular amino acid at the mutant position and/or corresponding wide-type position in the effected gene. Peptides containing cancer antigens are optionally tested for binding to HLA antigens of the cancer patient. Peptides containing the cancer antigens are evaluated for activating cytotoxic T lymphocytes (CTL) cell lines from the cancer patient or from an HLA-matched donor. The cancer antigen(s) identified for a cancer patient are used to prepare a cancer vaccine and to treat the cancer patient.
