An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction

March 24, 2020

Title

An Immune-Stimulatory Helix–Loop–Helix Peptide: Selective Inhibition of CTLA-4–B7 Interaction

Author

Tharanga M.R. Ramanayake Mudiyanselage, Masataka Michigami, Zhengmao Ye, Atsuko Uyeda, Norimitsu Inoue, Kikuya Sugiura, Ikuo Fujii, Daisuke Fujiwara

Year

2020

Journal

ACS Chemical Biology

Abstract

Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix–loop–helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4–B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a KD of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4–B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4–B7-1 interaction with an IC50 of 1.1 ± 0.03 μM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.

Instrument

J-820

Keywords

Circular dichroism, Secondary structure, Biochemistry