Inhibition of PD1:PD-L1 interaction by an E. coli-derived optimized PD1 variant

• Published in: Biochemical and biophysical research communications Vol. 506; no. 3; pp. 731 - 738
• Main Authors: Brand Shwartz, Michal, Assor, Mayan, Dotan, Nesly, Ratzon, Einav, Cohen, Elad, Ruimi, Nili, Bloch, Itai, Gal, Maayan, Yadid, Itamar
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 30.11.2018
• Subjects: 60 APPLIED LIFE SCIENCES; Amino Acid Sequence; Animals; APOPTOSIS; B7-H1 Antigen - metabolism; CELL PROLIFERATION; CHO Cells; Cricetinae; Cricetulus; Directed evolution; E. coli-based ELISA; ENZYME IMMUNOASSAY; Escherichia coli - metabolism; Gene Library; Humans; NEOPLASMS; PD1:PD-L1 interaction; Programmed Cell Death 1 Receptor - chemistry; Programmed Cell Death 1 Receptor - metabolism; Protein Binding; Protein Isoforms - chemistry; Protein Isoforms - metabolism; Protein optimization; RECEPTORS; Recombinant Proteins - metabolism; Reproducibility of Results; Solubility; Soluble receptors; Directed evolution; Soluble receptors; PD1:PD-L1 interaction; E. coli-based ELISA; Protein optimization
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2018.10.147

Immune-checkpoint receptors are a set of signal transduction proteins that can stimulate or inhibit specific anti-tumor responses. It is well established that cancer cells interact with different immune checkpoints to shut down T-cell response, thereby enabling cancer proliferation. Given the importance of immune checkpoint receptors, a structure-function analysis of these systems is imperative. However, recombinant expression and purification of these membrane originated proteins is still a challenge. Therefore, many attempts are being made to improve their expression and solubility while preserving their biological relevance. For this purpose, we designed an E. coli-based optimization system that enables the acquisition of mutations that increases protein solubility and affinity towards its native ligand, while maintaining biological activity. Here we focused on the well-characterized extracellular domain of the 'programmed cell death protein 1' (PD1), an immune checkpoint receptor known to inhibit T-cell proliferation by interacting with its ligands PD-L1 and PD-L2. The simple ELISA-based screening system shown here enabled the identification of high-affinity, highly soluble, functional variants derived from the extracellular domain of human PD1. The system was based on the expression of a GST-tagged variants library in E. coli, which enabled the selection of improved PD1 variants after a single optimization round. Within only two screening rounds, the most active variant showed a 5-fold higher affinity and 2.4-fold enhanced cellular activity as compared to the wild type protein. This scheme can be translated toward other types of challenging receptors toward development of research tools or alternative therapeutics. [Display omitted] •Cancer cells interact with immune checkpoints to shut down T cell response.•An optimization system for the selection of modified immune checkpoint proteins.•Using the described system improved PD1 variants were identified.•The improved variants are active in a cellular assay that mimics T cell:cancer interaction.