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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 linker, were 313x and 135x more potent in enhancing T-cell receptor (TCR) signaling in PD-1POS cells than monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169x and 35x 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 K2-based immune checkpoint medicines, and highlights the benefit of an IgG1 Fc-fusion to K2 that gains bivalency, effector functions and efficacy.
Original languageEnglish
Article number10
Pages (from-to)172-182
Number of pages11
JournalMolecular Therapy: Methods & Clinical Development
Issue number9
Publication statusPublished - 10 Sep 2021

Bibliographical note

© 2021 The Author(s).


  • Cancer
  • Immunotherapy
  • PD-L1


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