Inhibition of PD1:PD-L1 interaction by an E. coli-derived optimized PD1 variant
Michal Brand Shwartz1, Mayan Assor2, Nesly Dotan2
1Biochemistry Department, MIGAL -Galilee Research Institute, Kiryat-Shmona, 11016, Israel.
Abstract:
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.
Insights
Researchers developed an E. coli system to create improved versions of the programmed cell death protein 1 (PD1) immune checkpoint receptor. This system enhances protein solubility and affinity, aiding cancer research and therapeutics.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Immune checkpoint receptors regulate anti-tumor responses, but cancer cells exploit them to evade T-cell-mediated immunity.
- Recombinant expression and purification of these membrane proteins present significant challenges for structure-function analysis.
- Improving protein solubility and affinity while maintaining biological activity is crucial for studying immune checkpoints.
Purpose of the Study:
- To develop an E. coli-based system for optimizing immune checkpoint receptor variants.
- To enhance the solubility, affinity, and biological activity of the programmed cell death protein 1 (PD1) extracellular domain.
- To create a screening platform for identifying improved receptor variants for research and therapeutic applications.
Main Methods:
- Designed an E. coli-based system for directed evolution of immune checkpoint receptors.
- Expressed a library of glutathione S-transferase (GST)-tagged PD1 extracellular domain variants in E. coli.
- Utilized an enzyme-linked immunosorbent assay (ELISA)-based screening to select for high-affinity, soluble PD1 variants.
- Assessed cellular activity of selected variants compared to wild-type PD1.
Main Results:
- Successfully generated a library of PD1 extracellular domain variants in E. coli.
- Identified improved PD1 variants with significantly enhanced affinity and solubility after one optimization round.
- The most active variant exhibited a 5-fold increase in affinity and 2.4-fold enhanced cellular activity compared to wild-type PD1.
- Demonstrated the efficacy of the ELISA-based screening system for rapid variant selection.
Conclusions:
- The developed E. coli system effectively improves the expression, solubility, and affinity of PD1 extracellular domain variants.
- This platform facilitates the rapid identification of functional, high-affinity immune checkpoint receptor variants.
- The methodology is translatable to other challenging membrane proteins for developing novel research tools and therapeutics.
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