A Peptide Inhibitor against Human CTLA-4 and B7-1 Receptor Interaction.

Abstract

Cytotoxic T lymphocyte antigen-4 (CTLA-4) which is expressed on T-cells plays a crucial role in down regulation of the cancer immune response, and inhibitors of this immune check point can be used for treating cancer cells by stimulating the host immune system. Conformationally constrained polypeptides with high selectivity, efficacy, in vivo stability, and safety are promising alternatives to antibodies. Here, a selective inhibitor against human CTLA-4 and B7 receptor interaction was identified from improving the binding affinity of previously identified Helix-Loop-Helix (HLH) peptide, using error prone PCR method. Yeast surface-displayed HLH peptide library was constructed using CTLA-4 binding peptide from error prone PCR method and screened against human CTLA-4Ig by using magnetic activated cell sorting (MACS) and flowcytometry. One round MACS and three rounds of fluorescent activated cell sorting (FACS) were performed. The individual clones were identified by DNA sequencing. The screening conditions were improved in each round to have more potent binders. With each round of selection, CTLA-4 binding clones were enriched. Among the enriched clones, eight different clones were identified with affinities below 10 nM with frequencies of either 21% or 7.4%. One of the identified peptide sequences was chemically synthesised. The peptide showed higher binding affinity to CTLA-4, KD = 56.8 nM in surface plasmon resonance (SPR) in a direct binding assay. Further, the peptide inhibited the interaction between h-CTLA-4 and h.B7-1 with IC50 of 1.6 μM. Since CTLA-4 is a critical immune checkpoint that restricts the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.