Abstract The initial success with checkpoint blocking antibodies has evolved into a tremendous effort in combination therapy to further improve outcome in responsive cancers and to overcome resistance. There is strong rationale for this approach, as the cancer-immunity cycle has many points for intervention, and the immune system has developed multiple pathways to the same end. The use of bispecific antibodies provides opportunities to engage two pathways simultaneously that can result in greater efficacy, improved safety profile, and simplified development compared to separately administered antibodies. To generate bispecific antibodies that blocked the PD-1 signaling pathway, we developed and characterized fully human PD-L1 specific antibodies generated using human Ig transgenic mice. A lead antibody was chosen for its potent activity in functional assays and good manufacturing properties. Our initial bispecific construct couples our CD27 agonist antibody with the PD-L1 antibody (αCD27xαPD-L1) as we have previously shown that this combination led to improved therapeutic activity in the BCL1 disseminated lymphoma model. We found that the αCD27xαPD-L1 was more potent in CD27 costimulation assays such as NFkB activation, and T cell cytokine production compared to the parental antibodies. Similarly, αCD27xαPD-L1 was more efficacious in vivo as measured by Ag-specific T cell IFN-γ production and antitumor activity. Importantly, the bispecific antibody was more effective than the combination of anti-CD27 and anti-PD-L1 antibodies in the BCL1 lymphoma model. The enhanced efficacy was attributed to more efficient cross-linking of the bispecific antibody/CD27 receptor, resulting in stronger T cell activation. In addition, we observed that CD27 signaling increased PD-L1 expression on both tumor and immune cells, which may further enhance the activity of the bispecific antibody. The use of bispecific antibodies can improve the efficacy and profile of immunotherapeutic monoclonal antibodies by combining complementary functionalities and enhancing target cross-linking in the relevant tissues. We are currently exploring this approach with multiple antibodies from our pipeline. Citation Format: Laura A. Vitale, Lawrence J. Thomas, Li-Zhen He, Thomas O'Neill, Jenifer Widger, Andrea Crocker, James Testa, Karuna Sundarapandiyan, Eric Forsberg, James Boyer, James Storey, Joel Goldstein, Henry C. Marsh, Tibor Keler. Development of novel bispecific immune modulating antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5624.