Background: Neoadjuvant therapy is now a standard strategy for localized PDAC, and this preoperative window provides an excellent opportunity in which to test novel therapeutic approaches. Trials using IO in PDAC have largely been unsuccessful, and immune tolerance is implicated as a major mechanism of IO resistance. The gut and tumor microbiome have emerged as key modulators of response to both IO and chemotherapy. High tumor microbial diversity has been linked to longer survival in PDAC, and gut microbiota may have the ability to colonize pancreatic tumors. There is preclinical evidence that endogenous microbiota promotes the immunosuppressive tumor microenvironment characteristic of PDAC through stimulation of pro-tumor regulatory T cells and myeloid-derived suppressor cells at the expense of anti-tumor activated CD4+ and CD8+ T cells. Further, preclinical data show that ablation of the gut microbiota may induce T cell activation, improve immune surveillance, and increase sensitivity to IO. We hypothesize that ablative antibiotics (abx) will activate tumor infiltrating T cells and enhance IO activity in PDAC. Methods: This is a multi-center, single-arm, open-label pilot study of pre-operative chemotherapy followed by abx and pembrolizumab to evaluate overall immune response to abx + IO. Eligible patients will have histologically confirmed, resectable PDAC, without probiotic consumption or use of immunosuppressive agents. Patients will be enrolled at diagnosis after undergoing a baseline biopsy. They will then receive mFOLFIRINOX every 2 weeks for 5 cycles. After completion of chemotherapy, ciprofloxacin 500 mg PO BID and metronidazole 500 mg PO TID will be administered for 21 days, and pembrolizumab 200 mg IV x1 will be given 7 days after initiation of abx. Patients will then undergo surgical resection and adjuvant therapy at the investigators’ discretion. On-treatment biopsy will be obtained prior to cycle 5 of mFOLFIRINOX. Blood and stool will be collected at baseline, during mFOLFIRINOX therapy, before and after pembrolizumab administration, and postoperatively. The primary endpoint is the overall immune response, which will be measured as activation of one or more of the T cell markers HLA-DR, CD38, CD25, Ki67, and CD69, defined as an increase in expression level of at least 20% from the on-treatment specimen to the surgical specimen, before and after abx + IO. Key secondary endpoints will be the evaluation of adverse events, R0 resection rate, histologic regression score, objective response rate, and overall survival rate. Correlative studies will be carried out to evaluate immune and microbiome changes in the blood and tissue following abx and pembrolizumab. These findings will be correlated with clinical endpoints. The target study accrual is 25 patients. Clinical trial information: NCT05462496.