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

General audience description: Cancer cells use a protein called PD-L1 to paralyze immune cells with cancer killing abilities. We enhanced the potency of K2, a fragment of camel antibodies that binds PD-L1 and as such inhibits PD-L1’s immune paralyzing effects.

Abstract: 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 313- and 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 169- and 35-fold 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.