A Conformationally Constrained Peptide Binder against Human Cytotoxic T-lymphocyte Antigen-4 (CTLA-4)

Abstract

Immune checkpoint blockade enhances T cell activation against cancer cells. The HLH peptide is a molecular scaffold that can be used to generate molecular-targeting therapeutics. Hence, a conformationally constrained peptide against a crucial immune checkpoint, human (h) cytotoxic T-lymphocyte Antigen-4 (CTLA-4) was generated by screening a phage library of helix-loop-helix (HLH) peptides by bio-panning the target protein which was immobilized on ELISA plates; as a control experiment, the library was screened against human IgG. The bio-panning was repeated with five rounds, and the conditions were improved in each round. The number of the binding phage clones were increased from first to fourth rounds of bio-panning. The binding phage clones were enriched 6 times compared to the control binding phage clones from fourth to fifth round. Five phage clones (R4-7, R4-14, R4-15, R5-2, R5-6), which were randomly selected from the phage clones enriched in fourth and fifth rounds, and R4-14 showed specific binding affinity to h-CTLA-4-Ig. In direct binding assay, the binding affinity of thioredoxin (Trx) fused R4-14 was measured (KD = 82.6 M), while the control peptide, YT1-S-Trx showed no binding. The binding affinity of h-CTLA-4-Ig to its native counter-part h-B7-1 is stronger (KD=0.2μM-0.4μM) compared to the binding affinity of R4-14Trx to h-CTLA-4-Ig. Further, R4-14-Trx showed selective binding to h-CTLA-4 using ELISA, but no binding activity to the other proteins. Further improvements of R4-14 are necessary to generate more potent binder against CTLA-4 to inhibit the interaction between h-CTLA-4 and its native counter-part B7-1. Since CTLA-4 is a critical immune checkpoint, inhibitory HLH peptide may represent a new class of drug candidates for immunotherapy.