Background: Cancer vaccines may expand intratumoral CD8+ T cells, potentially increasing response rates to PD-1 blockade. SNS-301 is a bacteriophage-based cancer vaccine against aspartate β- hydroxylase (ASPH), a prevalent tumor-associated antigen. The objectives of this Phase 1/2 study were to evaluate the safety, immunogenicity and preliminary efficacy of SNS-301 plus pembrolizumab in patients (pts) not achieving tumor reductions on PD-1 blockade alone (Cohort A) or PD-1 blockade naïve patients (Cohort B). Methods: This trial enrolled SCCHN pts with locally advanced, unresectable or metastatic/recurrent ASPH⁺ tumors per screening immunohistochemistry. Intradermal SNS-301 (1x10¹¹ particles) + pembrolizumab (200mg IV q3w or 400mg IV q6w) was administered to pts with stable disease (SD) or unconfirmed progressive disease on ongoing PD-1 blockade of at least 12 weeks duration in Cohort A, and anti-PD1/PD-L1 naïve patients in Cohort B. Each cohort utilized a Simon Two Stage design with ORR assessed per RECIST1.1 at 12 weeks as the primary endpoint. The null hypothesis (H₀) was 5% ORR for Cohort A and 13.3% for cohort B. Enrollment of stage 2 was triggered if the ORR rate was ≥ 1 out of 15 initial evaluable patients (Cohort A) or ≥2 out of 15 (Cohort B). An initial “safety run-in” was instituted to ensure safety of the combination. Blood samples were collected to evaluate B and T cell responses using ELISA and ELISPOT assays, respectively. Results: Safety. No dose-limiting toxicities were identified in the “safety run-in” (initial 3 pts dosed). Overall, 25 pts were evaluable for safety assessment. 18 pts reported at least one treatment-emergent adverse event (TEAE), the majority of which were ≤ Grade 3. Grade 3 TRAEs included pruritus, rash, dehydration and QT prolongation. Three pts discontinued the study treatment due to TEAE and none were related to study treatment. There were three deaths reported during the follow-up period of the study and none were treatment related. Efficacy. 18 pts in Cohort A and 1 pt in Cohort B were evaluable for response per RECIST1.1 at 12 weeks. In Cohort A, one patient in Stage 1 had a PR, which triggered enrollment into Stage 2. In addition, 8 patients experienced SD as the best overall response. In Cohort B, a single PD-1/PD-L1 naïve patient was evaluable for efficacy and experienced a PR on the combination. Only a single pt in Cohort A experienced an AE of special interest (Grade 1 injection site pain). Pts treated with SNS-301 + pembrolizumab did not demonstrate significant ASPH-specific T-cell responses or increases in serum ASPH antibody titers. Conclusions: The combination of SNS-301 and pembrolizumab was well-tolerated with few treatment-related TEAEs. Importantly, pts treated with SNS-301 + pembrolizumab did not demonstrate ASPH-specific antibody or T-cell responses. Based on insufficient immunologic and clinical activity, the trial was terminated early. Clinical trial information: NCT04034225.