Formatting and gene-based delivery of a human PD-L1 single domain antibody for immune checkpoint blockade

Robin Maximilian Awad; Quentin Lecocq; Katty Zeven; Thomas Ertveldt; Lien De Beck; Hannelore Ceuppens; Katrijn Broos; Yannick De Vlaeminck; Cleo Goyvaerts; Magali Verdonck; Geert Raes; Alexander Van Parys; Anje Cauwels; Marleen Keyaerts; Nick Devoogdt; Karine Breckpot

doi:10.1016/j.omtm.2021.05.017

Formatting and gene-based delivery of a human PD-L1 single domain antibody for immune checkpoint blockade

Mol Ther Methods Clin Dev. 2021 Jun 4:22:172-182. doi: 10.1016/j.omtm.2021.05.017. eCollection 2021 Sep 10.

Authors

Robin Maximilian Awad¹, Quentin Lecocq¹, Katty Zeven^{1

2}, Thomas Ertveldt¹, Lien De Beck¹, Hannelore Ceuppens¹, Katrijn Broos¹, Yannick De Vlaeminck¹, Cleo Goyvaerts¹, Magali Verdonck¹, Geert Raes^{3

4}, Alexander Van Parys⁵, Anje Cauwels⁵, Marleen Keyaerts^{2

6}, Nick Devoogdt², Karine Breckpot¹

Affiliations

¹ Laboratory for Molecular and Cellular Therapy, Department of Biomedical Sciences, Vrije Universiteit Brussel, 1090 Brussels, Belgium.
² In Vivo Cellular and Molecular Imaging Laboratory, Department of Medical Imaging, Vrije Universiteit Brussel, 1090 Brussels, Belgium.
³ Laboratory of Cellular and Molecular Immunology, Department of Bioengineering Sciences, Vrije Universiteit Brussel, 1050 Brussels, Belgium.
⁴ Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, 1050 Brussels, Belgium.
⁵ Cytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB Medical Biotechnology Center, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium.
⁶ Nuclear Medicine Department, UZ Brussel, 1090 Brussels, Belgium.

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-1^POS 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.

Keywords: gene therapy; nanobody; programmed death-ligand 1; single domain antibody.