Background: EphB4 receptor tyrosine kinase and its ligand EphrinB2 are highly induced in head neck squamous cell carcinoma (HN SCC) tumor cells and vessels, particularly in HPV negative tumors. Each are predictors for poor survival with worse prognosis when both are induced. EphB4 provides tumor cell survival and EphrinB2 inhibits immune cell invasion. Soluble EphB4-Alb blocks bidirectional signaling, enhances immune cell recruitment alone and when combined with PD-1 antibody. Methods: A phase II trial of sEphB4-Alb combined with pembrolizumab accrued HN SCC patients after failure of one or more prior regimens. IHC positivity for p16 was used as a surrogate for HPV infection. Treatment regimen was sEphB4-Alb 10 mg/kg weekly IV infusion with pembrolizumab 200 mg IV infusion every three weeks. Study endpoints were toxicity, overall response rates (ORR) and overall survival (OS). Response to therapy was based on RECIST 1.1 criteria. Patient tumor samples were collected at baseline with a 2^nd biopsy at week 8 on therapy, for tissue analysis of PD-L1, EphrinB2 and other biomarkers. Results: Twenty-four patients were accrued to the phase II trial combination of sEphB4-Alb and pembrolizumab. Age, sex, prior treatment, HPV status, and response data are summarized in the table below. The most common toxicity was hypertension with 8 patients experiencing grade 3 HTN. No grade 4 or above toxicities were observed. Among HPV negative cases, partial and complete responses were observed in 6 of 14 patients (43%) with complete response (CR) observed in 3 of 6 responders. Additionally, rapid response was observed in 3 of 14 HPV negative patients. Response was associated with increase in immune markers on 2^nd biopsy. Median overall and progression-free survival in all patients was 12.6 months and 8.6 months, respectively. Conclusions: 1. sEphB4-Alb was well tolerated in combination with PD-1 antibody. 2. sEphB4-Alb was associated with increased immune response to tumor, when combined with PD-1 antibody. 3. sEphB4-Alb appears to have substantial activity (including complete remission) when combined with PD-1 antibody in relapsed/refractory HPV negative HN SCC. Clinical trial information: NCT03049618