A nalysis of spontaneous immune responses against cancer in humans has led to the identification of a large number of tumor antigens (1). The majority of these antigens can be classified into one of the following categories according to their expression pattern, function, or origin: cancer-testis (CT) antigens, e.g., MAGE (2, 3) and NY-ESO-1 (4), which are aberrantly expressed in tumor cells but that, with the exception of germ cells, are silent in normal cells; differentiation antigens of the melanocyte lineage, e.g., Melan A͞MART-1 (5, 6), tyrosinase (7), and gp100 (8, 9); mutational antigens, e.g., MUM-1 (10), p53 (11, 12), and CDK4 (13); overexpressed ''self'' antigens, e.g., HER2͞neu (14) and p53 (12); and viral antigens, e.g., HPV (15) and EBV (16). Spontaneous immune responses elicited by these antigens are either predominantly cellular, e.g., tyrosinase (17, 18) and Melan A͞MART-1 (9, 19), or are associated with a strong humoral immune component, e.g., NY-ESO-1 (20) and p53 (12).NY-ESO-1 is a highly immunogenic CT antigen, inducing simultaneous cellular and humoral immune responses in a high percentage of patients with advanced NY-ESO-1-expressing tumors (20,21). Detectable NY-ESO-1 serum antibody depends on the presence of NY-ESO-1-expressing tumor, and antibody titers correlate with the clinical development of disease (20,22). NY-ESO-1-specific CD8ϩ T-cell responses were detected in more than 90% of NY-ESO-1 antibody-positive patients, whereas NY-ESO-1 antibody-negative patients showed no detectable NY-ESO-1-specific T-cell reactivity (23).The present study was initiated to evaluate the effects of active immunization with NY-ESO-1 peptides in NY-ESO-1 antibodynegative and -positive patients. Three naturally processed NY-ESO-1 peptides presented by HLA-A2 were used for intradermal immunization, first alone and then in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) as a systemic adjuvant. The following parameters were monitored in this trial: (i) peptide-specific CD8ϩ T-cell responses; (ii) delayed-type hypersensitivity (DTH) reactivity; (iii) NY-ESO-1-specific antibody responses; and (iv) disease status. MethodsImmunization Protocol. Twelve HLA-A2ϩ patients with progressing NY-ESO-1-expressing metastatic tumors of different types and meeting predefined entry criteria were selected for immunization in the LUD97-008 protocol sponsored by the Ludwig Institute for Cancer Research. Immunizations were performed with three HLA-A2-binding NY-ESO-1 peptides derived from NY-ESO-1 and initially identified by the T-cell line NW38-IVS-1 (21). The NY-ESO-1 peptide sequences were: p157-167 (SLLM-WITQCFL), p157-165 (SLLMWITQC), and p155-163 (QLSLLMWIT). The HLA-A2-presented influenza matrix peptide p58-66 (GILGFVFTL) was used as a positive control for immune responses in vitro and in vivo. Peptides (Ͼ90% purity) were manufactured according to good manufactorial practice guidelines (Multiple Peptide Systems, San Diego) and solubilized in 100% DMSO. Intradermal injection of the 100% DMSO͞pepti...