Background: Preclinical studies suggest that MMP9 inhibition relieves immune suppression and promotes T cell infiltration to potentiate checkpoint blockade. To test this hypothesis, patient tumor samples obtained in a phase 2, open-label, randomized study (NCT02864381) of previously treated advanced gastric cancer comparing the MMP9-specific inhibitor andecaliximab (ADX) plus nivolumab (N) vs N alone were evaluated for T cell biomarkers. Methods: CD8 and PD-L1 (28-8 DAKO) were assessed by immunohistochemistry. IFNg, Teffector (Teff), and activated CD8+ T cell (ActT) gene signatures (GS) were assessed by RNASeq in archival baseline (BL) and biopsies obtained between weeks 5 and 9 (on-treatment; OT). Results: For both N and ADX/N, intratumoral CD8+ cells were significantly increased in OT biopsies relative to BL. The CD8+ OT increase was significantly greater for the ADX/N treatment relative to N in the PD-L1+ subgroup. In the ADX/N group only, IFNg, Teff, and ActT GS were significantly higher in OT biopsies relative to BL. OT change from BL of ≥ 300% vs <300% in CD8+ cells was associated with longer PFS (HR = 0.50, p = 0.032). The percentage of patients with increased CD8+ cells in OT biopsies was higher in the ADX/N arm. Conclusions: In the PD-L1+ ADX/N group, there was a significantly greater magnitude of CD8+ cell density increase, which was associated with gene signatures of T cell activation. More ADX/N-treated patients had an increase in tumor-associated CD8+ cells. Longer PFS was observed for patients in which CD8+ cells increased by ≥ 300%. These results are consistent with the hypothesis that ADX potentiates checkpoint inhibition by favorably altering the tumor immune microenvironment. Clinical trial information: NCT02864381

*PD-L1+: ≥1% tumor cells and associated immune cells; ^†nominal between arms ns: no GS was significant