Directed Evolution of PD-L1-Targeted Affibodies by mRNA Display

Abstract

Therapeutic monoclonal antibodies directed against PD-L1 (e.g., atezolizumab) disrupt PD-L1:PD-1 signaling and reactivate exhausted cytotoxic T-cells in the tumor compartment. Although anti-PD-L1 antibodies are successful as immune checkpoint inhibitor (ICI) therapeutics, there is still a pressing need to develop high-affinity, low-molecular-weight ligands for molecular imaging and diagnostic applications. Affibodies are small polypeptides (∼60 amino acids) that provide a stable molecular scaffold from which to evolve high-affinity ligands. Despite its proven utility in the development of imaging probes, this scaffold has never been optimized for use in mRNA display, a powerful in vitro selection platform incorporating high library diversity, unnatural amino acids, and chemical modification. In this manuscript, we describe the selection of a PD-L1-binding affibody by mRNA display. Following randomization of the 13 amino acids that define the binding interface of the well-described Her2 affibody, the resulting library was selected against recombinant human PD-L1 (hPD-L1). After four rounds, the enriched library was split and selected against either hPD-L1 or the mouse ortholog (mPD-L1). The dual target selection resulted in the identification of a human/mouse cross-reactive PD-L1 affibody (M1) with low nanomolar affinity for both targets. The M1 affibody bound with similar affinity to mPD-L1 and hPD-L1 expressed on the cell surface and inhibited signaling through the PD-L1:PD-1 axis at low micromolar concentrations in a cell-based functional assay. In vivo optical imaging with M1-Cy5 in an immune-competent mouse model of lymphoma revealed significant tumor uptake relative to a Cy5-conjugated Her2 affibody.

Integrating Dynamic Network Analysis with AI for Enhanced Epitope Prediction in PD-L1:Affibody Interactions

“…[6][7][8] Such non-antibody sca↵old proteins can be engineered using in vitro directed evolution to isolate high a nity binding domains against a range of molecular targets. [9][10][11] With a molecular weight of ⇠6.5 kDa, the highly stable a body triple ↵-helix bundle 12,13 ( Fig. 1C) in particular o↵ers a promising alternative sca↵old.…”

Generation of human TMEM16F-specific affibodies using purified TMEM16F

“…Since the affibodies tested in this study were not the optimized affibodies, the binding affinity and inhibition could be increased by conducting maturation processes in the future study ( Grindel et al, 2022 ). Further, if we have the complex structure of TMEM16F-affibody, the affibodies could be engineered to enhance the binding affinity based on the findings on key residues for the affibody binding to TMEM16F.…”

High‐throughput screening and biological display technology: Applications in molecular imaging

“…The M1 affibody can also inhibit the PD-L1 signaling pathway at micromolar concentrations, further demonstrating its therapeutic potential. Millward and coworkers also selected the HER2 affibody and autophagy protein LC3 targeting ligands [83].…”