Abstract
Monoclonal antibodies that target the inhibitory immune checkpoint axis consisting of programmed cell death protein 1 (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 12 GS (glycine-serine) linker, were 313- and 135-fold more potent in enhancing T cell receptor (TCR) signaling in PD-1POS cells than was monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169- and 35-fold less potent in enhancing TCR signaling than was 2K2. 2K2 enhanced tumor cell killing in a 3D melanoma model, albeit to a lesser extent than avelumab. Therefore, an immunoglobulin (Ig)G1 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 K2-based immune checkpoint medicines, and it highlights the benefit of an IgG1 Fc fusion to K2 that gains bivalency, effector functions, and efficacy.
Highlights
Blockade of inhibitory immune checkpoints using monoclonal antibodies has proven clinical value in a variety of solid tumors and has become a mainstay therapy option.[1,2,3,4,5,6] Blockade of the inhibitory immune checkpoint consisting of programmed cell death protein 1 (PD-1, CD279) and its ligand PD-L1 (B7-H1, CD274) has received much attention, as this immune checkpoint hampers the antitumor immune response at multiple levels, i.e., the priming and effector phases.[7,8]PD-1 is a type 1 transmembrane receptor that is mainly expressed on the surface of T cells upon their activation.[9]
Restoration of T cell receptor (TCR) signaling upon interaction of PD-1POS T cells with PD-L1POS tumor cells requires high doses of K2-encoding blot
The lentiviral transfer plasmids were used for production of second-generation LVs that were quantified by measuring lentiviral vectors (LVs) the amount of reverse transcriptase (RT) (Figure S1B)
Summary
PD-1 is a type 1 transmembrane receptor that is mainly expressed on the surface of T cells upon their activation.[9] Engaging PD-1 on T cells interferes with T cell receptor (TCR) signaling, thereby preventing excessive stimulation, and maintaining tolerance to self-antigens.[10] This poses a barrier for antitumor immunity, as PD-L1 is highly expressed on dendritic cells that need to orchestrate the antitumor immune response.[11] In addition, PD-L1 is often abundantly expressed on immune cells and cancer cells in the tumor microenvironment (TME).[12,13,14] Expression of PD-L1 on the surface of cancer cells, myeloid cells, and lymphoid immune cells in the TME paralyzes the effector function of PD-1POS T cells. The PD-L1 signalosome in cancer cells promotes cancer cell survival through regulation of resistance to pro-apoptotic stimuli, such as interferons, thereby posing another barrier to antitumor immunity.[15]
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