895 Discovery of a safer 4–1BB agonist by targeting a membrane-proximal epitope combined with bispecific-mediated cross-bridging

Background Checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have paved the way for a new frontier of anti-cancer therapies that modulate our pre-existing immune system to fight against malignancies. 4-1BB is a tumor-necrosis superfamily member expressed on NK and T cells, that acts as a co-stimulatory receptor to improve effector/memory responses towards tumors. Early efforts have focused on the generation of agonist antibodies towards 4-1BB that relied on Fc-mediated cross-linking to cluster and induce receptor downstream signaling, but has led to liver- and immune-related toxicities. We have discovered a PD-L1 x 4-1BB bispecific that exhibits conditional agonist activity in the presence of PD-L1 with better safety features. Methods vHH binders to PD-L1 and 4-1BB were generated from immune libraries derived from camelids and selected via yeast display. Antibody screening was carried out by protein-protein interaction analysis and cell-based binding. Target-related activity was confirmed using luciferase reporter assays. Primary immune cells were also tested for T cell activation via the detection of IL-2 and IFNg secretion. PD-L1-mediated 4-1BB activation via cross-bridging was carried out using target cells expressing PD-L1 co-cultured with effector cells. X-ray crystallography was conducted to resolve the binding sites of both the anti-PD-L1 and anti-4-1BB vHHs. Both tumor efficacy and safety assessment were tested in human knockin mice. Results The 4-1BB binder of PM1003 was found to interact with the CRD4 domain of 4-1BB, as determined by X-ray crystallography. Binding to this domain does not affect the binding between 4-1BB and its ligand 4-1BBL. The anti-PD-L1 vHH binds to an epitope on PD-L1 that overlaps with the binding region of PD-1, and is thus effective in disrupting the interaction between PD-1 and PD-L1. Using luciferase reporter assays and primary cell assays we found the PM1003 could activate 4-1BB in the context of PD-L1-mediated cross-bridging. Data from human 4-1BB and PD-L1 knockin mice also showed that PM1003 could effectively control tumor growth without observing any toxicity signals. Conclusions PM1003 is a safe and efficacious bispecific antibody that blocks PD-L1 and concurrently activates 4-1BB receptor. An antibody with mild activity was selected directed against the CRD4 domain of 4-1BB to elicit effective potency while minimizing potential toxicity issues. This was reflected in our results. Thus, PM1003 is a potential next generation therapeutic antibody in the IO space that combines and synergizes two independent signaling pathways to control tumor growth.