Antibody blockade of semaphorin 4D to sensitize pancreatic cancer to immune checkpoint blockade

26 Background: Pancreatic ductal adenocarcinoma (PDAC) carries a dismal prognosis, and will soon become the second leading cause of cancer mortality. Unfortunately, T-cell directed immunotherapies have failed to demonstrate efficacy in PDAC. These failures may in part be mediated by an immunosuppressive tumor microenvironment (TME). Semaphorin 4D (Sema4D) is a glycoprotein which binds its cognate receptors Plexin B1/B2. Here we present our work in blocking Sema4D in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors and confirmed for disease by ultrasound. Mice were treated with FOLFIRINOX (5-FU, Irinotecan, Oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-Sema4D mAB (bi-weekly). Human and mouse circulating and tumor infiltrating leukocytes were interrogated through flow cytometry (FACS) for immune subset and expression of Sema4D and Plexin receptors. Archived human PDAC tissues were assessed through quantitative immunohistochemistry (IHC) for presence of Sema4D positive infiltrate. Results: Both FACS and IHC analysis of human PDAC specimens confirm the presence and increased prevalence over normal pancreata of Sema4D lymphocytes and Plexin B1/B2 expressing tumor associated macrophages (TAMs). KP2 orthotopically injected mice exhibited longer survival when treated with the triple combination of FOLFIRINOX, ICB, and anti-Sema4D antibody, compared to FOLFIRINOX alone, FOLFIRINOX plus ICB, or FOLFIRINOX plus anti-Sema4D antibody (P < 0.02). Flow cytometric analysis of anti-Sema4D and ICB treated murine tumors show a doubling of penetration by CD 8+ effector T cells within tumors compared to control groups (P = 0.03). A loss in Sema4D fluorescence signal via FACS in tumor-infiltrating CD3+ leukocytes was observed in mice treated with anti-SEMA4D, confirming penetration and target blockade within the TME. Conclusions: Sema4D and Plexin B1/B2 leukocytes penetrate human PDAC tumors, and treatment with Sema4D blocking antibody improved response to ICB in combination with standard of care FOLFIRINOX in preclinical murine studies.