Background: Based on the exploratory analysis of our previous studies of peptide vaccine, we concluded that the combination of adjuvants hLAG-3Ig + Poly-ICLC is essential for controlling negative immune checkpoints and enhancing the induction of CTLs to overcome the traditional peptide studies. Another issue with peptide vaccines is human leukocyte antigen (HLA) restriction. Hence, we developed novel multi-HLA-binding peptides derived from the tumor antigens, HSP70 and GPC3, and confirmed the high expression in many types of cancer. Methods: For the identification of peptides, HSP70- and GPC3-derived peptides that have high binding affinity to each of HLA-A2402, 0201, and 0206 were selected as candidate peptides by a binding prediction system (NEC Corporation). We then identified priority candidate peptides by using a peptide-binding assay. Using peripheral mononuclear blood cells from cancer patients, CD8+ T lymphocytes were stimulated with the candidate peptides, and an enzyme-linked immunospot assay was performed. Finally, we identified HSP70- and GPC3-peptide. In this phase I study of a novel peptide cancer vaccine for metastatic solid cancer, primary objective was to evaluate its safety and toxicity. Secondary objective was to examine the immune and clinical response, and also to determine the recommendable dose. This study used a three-tiered dose-escalation strategy with 3 patients’ cohorts. In addition to the 3 scheduled cases, 3 more cases were added and 6 cases were enrolled at the recommended dose. Results: Twelve HLA-A*24:02-, 02:01-, and 02:06-matched patients (esophagus, 3; colon, 4; liver, 3; pancreas, 1; stomach, 1) were treated in this study. No severe adverse effects related to the treatment were encountered. Peptide-specific CTL induction with HSP70 and GPC3 was observed in 10 and 11 patients, respectively. We observed decreased tumor marker expression in 6 cases, and disease control was observed in f5 patients (4, 3, 8, 2, 2 months, respectively). Conclusions: The combination cancer vaccine therapy using multi-HLA-restricted peptides and hLAG-3Ig + Poly-ICLC was safe and effective for treating solid tumors; it therefore warrants further clinical studies. Clinical trial information: UMIN000020440.