Optimized PD-1 Variants as non-antibody Biologic Drug

Immune-checkpoint receptors are a set of signal transduction proteins that can stimulate or inhibit specific anti-tumor response. The `programmed cell death 1` (PD1) protein is an immune checkpoint receptor expressed on the surface of activated T cells where it is known to modulate T-cells response during events such as pregnancy, tissue allografts, and autoimmune diseases. In cancer, PD-1 ligands (PDL-1/2) are over expressed on tumor cells and bind to PD-1 leading an immune-suppression. In this way, tumor cells evade antitumor response and facilitate their survival. Several mAbs were approved for the treatment of various cancer by targeting PD-1. However, there are several disadvantages that are associated to antibody drugs, such as production cost, stability, high molecular weight, Immunogenicity and Toxicity. The present study aims to interfere with the interactions between PD-1 to PD-L1 by designing an optimized PD-1 protein variant, using directed-evolution. For that purpose, the use of E. coli as the host organism is robust and cost-effective choice. In addition, it enables large flexibility to address different structure-function aspects regarding the target protein. We used a simple ELISA-based screening system that given a pair of interacting proteins, enable the selection of variant with better solubility and affinity. The system is based on the expression of PD-1 variants library in E.coli conjugated to GST-tag and on the binding strength of the variants to the ligand, PDL-1. 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. These results thus highlight the potential of those PD-1 variants as a small, non-antibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.