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

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Therapeutic monoclonal antibodies targeting PD-L1 are effective immune checkpoint inhibitors.
• There is a need for high-affinity, low-molecular-weight ligands for PD-L1 molecular imaging and diagnostics.
• Affibody scaffolds offer a stable molecular base for evolving high-affinity ligands.

Purpose of the Study:

• To select a PD-L1-binding affibody using mRNA display technology.
• To optimize the affibody scaffold for mRNA display, incorporating high library diversity and unnatural amino acids.
• To develop a novel imaging probe for PD-L1.

Main Methods:

• Randomization of the binding interface of a known affibody scaffold.
• Selection of the affibody library against recombinant human and mouse PD-L1 using mRNA display.
• Characterization of binding affinity, cell-surface binding, and functional inhibition of PD-L1:PD-1 signaling.
• In vivo optical imaging in a mouse lymphoma model.

Main Results:

• Identification of a human/mouse cross-reactive PD-L1 affibody (M1) with low nanomolar affinity.
• M1 affibody demonstrated similar binding affinity to cell-surface expressed hPD-L1 and mPD-L1.
• The M1 affibody inhibited PD-L1:PD-1 axis signaling in a cell-based assay.
• In vivo imaging showed significant tumor uptake of M1-Cy5 in a mouse lymphoma model.

Conclusions:

• mRNA display is a viable platform for selecting PD-L1-binding affibodies.
• The M1 affibody is a high-affinity, cross-reactive ligand with potential for PD-L1 diagnostic imaging.
• This affibody represents a promising low-molecular-weight alternative to antibodies for PD-L1 targeting.