Background: Complexes of Cholesteryl pullulan and NY-ESO-1 antigen (CHP-NY-ESO-1) present multiple epitope peptides to both the MHC class I and class II pathways. MIS416 is a non-toxic microparticle adjuvant that activates immune responses via NOD-2 and TLR9 pathways. Vaccination studies in mice have demonstrated that CHP-NY-ESO-1 with MIS416 enhanced anti-tumor efficacy. We conducted a First-in-Human clinical trial using CHP-NY-ESO-1 with adjuvant MIS416 for patients (pts) with NY-ESO-1-expressing refractory solid tumor (Clinical trial registration number, UMIN000005246, UMIN000008007). Methods: NY-ESO-1 expression was assessed by immunohistochemistry and/or RT-PCR. Pts received CHP-NY-ESO-1/MIS416 (C/M) 100/200, 200/200, 200/400 or 200/600 (μg/μg) (every 2 weeks, 6 doses total) followed by one vaccination every 4 weeks, until disease progression or unacceptable toxicity was observed. Response was assessed every 12 weeks by CT. The primary endpoints were safety and tolerability, and the secondary endpoint was immune response. Results: A total of 26 pts were enrolled. Median age was 70 years (range: 36-84). Median number of vaccination was 6 (range: 1-66). Seven pts (38%) received ≥7 doses. Enrollment to C/M 200/600 was stopped because of high frequency of SAE (2/4 pts) and little antitumor effect. Eight pts (31%) had SD or non-CR, non-PD. One pt (C/M 200/200) had no progression for 5 years at the end of study. Grade 1-2 injection site reaction was observed in all pts. Grade 3 drug-related AEs were observed in 6 pts (23%), hypertension (n = 5) and anorexia (n = 1). No grade 4-5 drug-related AEs were observed. Anti-NY-ESO-1 IgG1 response was attenuated significantly as the dose of MIS416 increased. Conclusions: C/M 200/200 and 200/400 were well tolerated. Further investigation is warranted. Clinical trial information: 000005246, 000008007.