Background: Stromal HA poses a physical barrier and protects tumor cells from immune surveillance. PEGPH20 is a pegylated, human recombinant PH20 hyaluronidase that remodels tumor stroma. Preclinical studies of PEGPH20 showed improved infiltration of cytotoxic T-lymphocytes and delivery of chemotherapy and PD1/PD-L1 antibodies. This study aimed to evaluate the efficacy, safety, and translational biomarkers of PEGPH20 plus pembrolizumab in pts with HA-high refractory mPC. Methods: mPC pts with HA-high expression, ECOG PS 0-1, ≤ 2 prior therapies for metastatic disease, life expectancy ≥ 12 weeks were treated with PEGPH20 3 µg/kg iv on D1, D8, D15 and pembrolizumab 200 mg iv on D1 in 21-day cycles. Primary endpoint was progression-free survival (PFS). Secondary endpoints were safety, overall survival (OS), and objective response rate (ORR). Blood and tumor biopsies were collected at baseline and on-study. Translational endpoints included flow cytometry and IHC for immune subsets, T-cell receptor sequencing, immune transcriptome, circulating cytokines, and plasma and tumor HA levels. Assuming a one-sided α-level of 0.05 and power of 80%, 31 evaluable pts were needed to detect an improvement of median PFS from 3 to 6 months. Results: Between May 2019 to Nov 2019, 38 pts were screened for HA expression, and 8 pts were enrolled, with median age 68 years (range 60-73), 7 males, and median 2 prior therapies (range 1-4). The accrual was stopped early by Halozyme Pharmaceuticals due to lack of benefit from PEGPH20 added to chemotherapy in the HALO-301 study. Treatment exposure median was 2 cycles (range 1-6). Reasons for study discontinuation were disease progression (n = 4), termination by sponsor (n = 3), patient withdrawal to enroll in hospice (n = 1). Treatment related toxicities were musculoskeletal (n = 6, grade 1/2), edema (n = 2, grade 1), fatigue (n = 1, grade 3), dyspnea (n = 1, grade 2), hypothyroidism (n = 1, grade 2). Median OS was 7.2 months (95% CI 1.2-11.8), and median PFS was 1.5 months (95% CI 0.9-4.4). Best response was stable disease (n = 2, 25%) lasting 2.2 and 9 months, respectively, and no responses were noted. Patients with available molecular sequencing data had MSS tumors. Translational biomarkers will be presented. Conclusions: Pembrolizumab and PEGPH20 did not increase PFS compared to historical data among heavily pretreated mPC pts, but the median OS of 7.2 months is encouraging. Translational analyses will provide insights into immune modulatory effects from PEGPH20 that could inform future studies with stroma targeted therapies and immune checkpoint blockade in mPC. Clinical trial information: NCT03634332.