Pancreatic ductal adenocarcinoma (PDAC), which accounts for more than 90% of the pancreatic cancer cases, unfortunately has an average 5-year survival rate of less than 10%. To date, both chemotherapy and immunotherapy, including the checkpoint inhibitors like PD-L1, CTLA-4, has not been effective in treating PDAC. Although PDAC is infiltrated with leukocytes, they are mostly the suppressive types such as myeloid- derived suppressor cells, regulatory B cells, T regulatory cells and M2-macrophages, likely due to elevated levels of molecules such as TGF-β that suppresses T-cell activation. We have identified Siglec-15 (S15), which can directly inhibit NFKβ/NFAT signaling resulting in suppressed T-cell proliferation and cytokine production, as a critical immune suppressor in tumor microenvironment. Interestingly, S15 is broadly upregulated in various cancers, including triple negative breast cancers and PDAC, as well as tumor-associated macrophages, with no detectible expression in other healthy cells besides monocytes. Elevated expression of S15 is correlated with poor survival in PDAC patients. Importantly, Siglec15 is highly expressed in PDAC cells with low PD-L1, suggesting Siglec15 may be involved in immune evasion observed in PDL1-negative PDAC. Given the overexpression of S15 in PDAC, we hypothesized that a bispecific antibody (bsAb) that binds both S15 and an immune-suppressive cytokine (TGF-β) could result in greater bsAb accumulation in the tumor, and thus synergistically enhances T-cell activation for improved tumor suppression. We hypothesize that bispecific T-cell engagers (BiTE) that bind S15 and CD3 could likewise enhance T-cell immunotherapy against PDAC, including breaking down the stromal barrier. In pilot studies, our S15/TGF-β bsAb afforded much more effective tumor suppression than a cocktail of anti-S15 and anti-TGF-β Ab in mouse model of triple negative breast cancer. Likewise, S15/CD3 BiTE effectively suppressed pancreatic tumor in a xenograft model. Building off these promising findings, we seek to explore whether simultaneously modulation of the tumor immune microenvironment with improved T-cell targeting can lead to more effective therapy against PDAC. Towards this goal, we will engineer in Aim 1 a panel of bsAb that binds S15 while possessing different number of binding domains against TGF-β. We will evaluate whether the increased number of TGF-β binding domains further reduce immune suppression in the tumor leading to more effective therapy in an orthotopic pancreatic model in mice. In Aim 2, we will investigate whether the most potent S15/TGF-β bsAb from Aim 1 may synergistically enhance S15/CD3 BiTE therapy, both in an orthotopic human pancreatic cancer model in NSG mice infused with human PBMC and in a syngeneic orthotopic mice pancreatic cancer model. If successful, our work may lead to improved treatment options for management of PDAC, as well as advance an overall framework to enhance immunotherapy against diffe...