Gene-based delivery of human PD-L1 single domain antibody formats for immune checkpoint blockade

Monoclonal antibodies that target the inhibitory immune checkpoint axis consisting of PD-1 and its ligand, PD-L1, have changed the immune-oncology field. We identified K2, an anti-human PD-L1 single domain antibody fragment, that can enhance T-cell activation and tumor cell killing. In this study, the potential of different K2 formats as immune checkpoint blocking medicines was evaluated using a gene-based delivery approach. We showed that 2K2 and 3K2, a bivalent and trivalent K2 format generated using a 12GS linker, were 313and 135-fold more potent in enhancing T-cell receptor (TCR) signaling in PD-1 positive cells than monovalent K2. We further showed that bivalent constructs generated using a 30GS linker or disulfide bond were 169and 35fold less potent in enhancing TCR signaling than 2K2. 2K2 enhanced tumor cell killing in a 3D melanoma model, albeit to a lesser extent than avelumab. Therefore, an IgG1 antibody-like fusion protein was generated, referred to as K2-Fc. K2-Fc was significantly better than avelumab in enhancing tumor cell killing in the 3D melanoma model. Overall, this study describes a new immune checkpoint medicine, and highlights the benefit of an IgG1 Fc-fusion to K2 that gains bivalency, effector functions and efficacy. REFERENCES In alphabetical order: Broos, Lecocq, Xavier, Bridoux, Nguyen, Corthals, et al. (2019). Evaluating a Single Domain Antibody Targeting Human PD-L1 as a Nuclear Imaging and Therapeutic Agent. Cancers 11: 872. Emeagi, PU, van Lint, S, Goyvaerts, C, Maenhout, S, Cauwels, A, McNeish, IA, et al. (2012). Proinflammatory characteristics of SMAC/DIABLO-induced cell death in antitumor therapy. Cancer Research 72: 1342–1352. Lecocq, Q, de Vlaeminck, Y, Hanssens, H, D’Huyvetter, M, Raes, G, Goyvaerts, C, et al. (2019). Theranostics in immuno-oncology using nanobody derivatives. Theranostics 9: 7772–7791. Milone, MC and O’Doherty, U (2018). Clinical use of lentiviral vectors. Leukemia 32: 1529–1541. Versteven, M, van den Bergh, JMJ, Broos, K, Fujiki, F, Campillo-Davo, D, de Reu, H, et al. (2018). A versatile T cell-based assay to assess therapeutic antigenspecific PD-1-targeted approaches. Oncotarget 9: 27797–27808.