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.
