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

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 169and 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.

Discovery of Anti-PD-L1 Human Domain Antibodies for Cancer Immunotherapy

“…PD-L1-specific blockade would be preferred to PD-1-specific blockade ( 19 ). Compared to traditional intact antibody, sdAb, the smallest naturally occurring antibody fragment in Camelid family, retains a comparable blocking ability as whole antibodies ( 15 ). sdAbs have numerous advantages, including small size, low immunogenicity, and easy production.…”

“…This limitation can be addressed by constructing a bivalent or trivalent form of sdAbs. For example, the bivalent and trivalent format of a sdAb are 313- and 135-fold more potent, respectively, than the monovalent format ( 15 ).…”

“…Another disadvantage of sdAbs is their monovalency which leads to relatively lower affinity and blocking efficiency compared to antibody-based checkpoint inhibitors. This limitation can be addressed by constructing a bivalent or trivalent form of sdAbs ( 15 ). sdAbs can be discovered through animal immunization or various display libraries, such as phage display, yeast display, bacterial display, and ribosome display ( 16 – 18 ).…”

A synthetic DNA template for fast manufacturing of versatile single epitope mRNA

“…From the same leukapheresis material, we stored the monocyte-depleted fraction as previously described. 74 …”

“…From the same leukapheresis material, we stored the monocyte-depleted fraction as previously described. 74 Generation of DNA templates for in vitro mRNA transcription…”