Background: Poly-ICLC is a synthetic dsRNA complex which directly activates DCs and also triggers NK cells to kill tumor cells. Compared to LPS and R848, Poly-ICLC is the most potent TLR adjuvant due to its induction of cytokines such as IL-12 in the absence of IL-10, and maintenance of high levels of CD80 and CD86 in DCs. This study assessed the therapeutic potential of TLR3 activation by adding Poly-ICLC to a NY-ESO-1 protein vaccine with and without Montanide in surgically resected stage IIB-IV melanoma patients. Methods: In Phase I of the study, patients received subcutaneous injection of 100μg NY-ESO-1 protein and 1.1mL Montanide emulsified in escalating doses of Poly-ICLC: 0.35mg (cohort 1; N=3), 0.70mg (cohort 2; N=3), or 1.4mg (cohort 3; N=3). The cycles of vaccination were repeated every 3 weeks for a total of 4 cycles. In Phase II of the study, patients were randomized to subcutaneous vaccination of 100μg NY-ESO-1 protein with 1.4 mg Poly-ICLC, the dose established in the Phase I of the study, (Arm A; N=12) or with 100μg NY-ESO-1 protein, 1.4mg Poly-ICLC and 1.1ml Montanide (Arm B; N=12). The cycles of vaccination were repeated every 3 weeks for a total of 4 cycles. Blood samples were collected at baseline, one week after each cycle of vaccination, and at follow-up visit for the assessment of NY-ESO-1-specific humoral and cellular immune responses. Clinical trial information: NCT01079741.