Abstract 7304: Cytotoxic PD-L1/PD-L2 dual-specific antibodies couple tumor stroma remodeling with checkpoint blockade to drive rejection of “cold” cancers

Abstract Introduction: Antibody blockade of either the Programmed Death 1 (PD-1) immune checkpoint receptor on T cells or its cognate ligand PD-L1 on tumor cells and stroma has revolutionized oncology but fails to regress “cold” tumors lacking T cell infiltration. We leveraged the iterative selection capacity of yeast-displayed fully human antibody selection to identify dual PD-L1/PD-L2 specific antibodies (DIPDL) that block their engagement of both PD-1 and B7-1. Finally, we engineered these DIPDL to efficiently kill PD-L1 and PD-L2 expressing tumors and stroma. Materials and Methods: Antibody binding to PD-Ligands was measured by Octet. The Promega PD-1 assay system was used to measure EC50s for reversal of PD-1 driven T cell inhibition. Retrogenix microarray technology was used to determine specificity of target binding. Surrogate and direct assays were used to measure ADCC and ADCP efficiency. Murine syngeneic tumor models were tested for PD-1 blockade versus DiPDL antibodies to mediate tumor regression. Flow cytometry was used to characterize changes in the tumor immune microenvironment. Human tumor xenografts were implanted into immunodeficient mice to study the cytotoxic potential of these antibodies. Results: We leveraged multiple antibody engineering technologies to generate dual PD-L1/PD-L2 specific antibodies capable of blocking ligand interactions with PD-1 and B7-1, and also mediating efficient tumor and stromal depletion. These Fc engineered DIPDL kill human PD-L1+ and/or PD-L2+ cells in vitro using both NK and myeloid effectors. In vivo studies of murine syngeneic tumor models revealed that these DIPDL antibodies significantly outperform PD-1 blockade across both “hot” and “cold” cancers. Tumor microenvironment analysis showed that myeloid stromal depletion correlates with T cell and dendritic cell expansion and therapeutic response. Lastly, treatment of immunodeficient mice bearing human triple negative breast cancer with these cytotoxic DIPDL significantly inhibited tumor growth and reduced myeloid stromal density. Conclusions: These DIPDL represent a new generation of immunotherapeutic which couple superior PD-1 pathway blockade with direct depletion of both immunosuppressive and T-cell exclusionary stroma and tumors. Across murine models, these DIPDL deliver transformative efficacy overcoming limitations of simple PD-1 blockade and have moved to an ongoing Phase I clinical trial. Citation Format: Ahmad Salameh, Coline Couillault, Anupallavi Srinivasamani, Qinying Liu, Elisabetta Bolli, Christine Gagliardi, Guillaume Trusz, Nadthakarn Boland, Paul Blezinger, Dongxing Zha, Manuela Iezzi, Bianka Prinz, Federica Cavallo, Federica Pericle, Michael A. Curran. Cytotoxic PD-L1/PD-L2 dual-specific antibodies couple tumor stroma remodeling with checkpoint blockade to drive rejection of “cold” cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7304.