Discovery of Cyclic Peptide Inhibitors Targeting PD-L1 for Cancer Immunotherapy

Abstract

Blockade of the interaction between programmed cell death ligand-1 (PD-L1) and its receptor PD-1 has shown great success in cancer immunotherapy. Peptides possess unique characteristics that give them significant advantages as immune checkpoint inhibitors. However, unfavorable physicochemical properties and proteolytic stability profiles limit the translation of bioactive peptides as therapeutic agents. Studies have revealed that cyclization improves the biological activity and stability of linear peptides. In this study, we report the use of macrocyclization scanning for the discovery of cyclic anti-PD-L1 peptides with improved bioactivity. The cyclic peptides demonstrated up to a 34-fold improvement in the PD-1/PD-L1 blocking activity and significant in vivo anti-tumor activity. Our results demonstrate that macrocyclization scanning is an effective way to improve the serum stability and bioactivity of the anti-PD-L1 linear peptide. This strategy can be employed in the optimization of other bioactive peptides, particularly those for protein-protein interaction modulation.

Figure 1.. Screening of anti-PD-L1 cyclic peptides.

(A) Sequences of cyclic peptides designed by macrocyclization scanning. (B) Normalized blocking efficiency for anti-PD-L1 cyclic peptides at 5 μM. Data are presented as mean ± SD (n=3). Chemical structures of cyclic peptides C7 (C) and C12 (D).

Figure 2.. Blockade efficiency of anti-PD-L1 cyclic peptides against the PD-1/PD-L1 interaction.

Blocking profiles of the peptides C7 (A), C12 (B), and CLP-2 (C) against the human PD-1/PD-L1 interaction. Blocking profiles of the peptides C7 (D), C12 (E), and CLP-2 (F) against the mouse PD-1/PD-L1 interaction. Data are presented as mean ± SD (n = 3).

Figure 3.. Competitive SPR to investigate the blocking activities of peptides against PD-1/PD-L1 interaction.

(A) Schematic representation of competitive SPR. SPR sensorgrams of the peptides C7 (B), C12 (C), and CLP-2 (D) at different concentrations. Blocking curves of the peptides C7 (E), C12 (F), and CLP-2 (G). Data are presented as mean ± SD (n = 3).

Figure 4.. Binding of anti-PD-L1 peptides to human PD-L1-overexpressing cells (MDA-MB-231 and DU145) and PD-L1-deficient cells (MDA-MB-231 PD-L1 knockout and MCF-7).

Binding of Cy5-labeled peptides C7 (A), C12 (B), and CLP-2 (C) to MDA-MB-231 and MDA-MB-231 PD-L1 knockout cells. Binding of Cy5-labeled peptides C7 (D), C12 (E), and CLP-2 (F) to DU145 and MCF-7 cells. Data are presented as mean ± SD (n=3). (* p < 0.05; ** p < 0.01)

Figure 5.. Cyclic anti-PD-L1 peptides reinvigorate PBMCs co-cultured with DU145 cell.

(A) Annexin V-FITC/PI flow cytometry analysis of PBMCs co-cultured with DU145 cells in the presence of anti-PD-L1 peptides (10 μM) or antibody (2 μM). (B) Percentage of apoptotic PBMCs co-cultured with DU145 cells in the presence of anti-PD-L1 peptides (10 μM) or antibody (2 μM). Flow cytometry analysis (C) and bar graphs (D) showing percentage of CD8⁺/IFN-γ⁺ T cells from PBMCs co-cultured with DU145. Flow cytometry analysis (E) and bar graphs (F) showing percentage of CD4⁺/IFN-γ⁺ T cells from PBMCs co-cultured with DU145. Data are presented as mean ± SD (n = 3). (* p < 0.05; ** p < 0.01)

Figure 6.. Cytotoxicity and serum stability of cyclic anti-PD-L1 peptides.

(A) Cytotoxicity of peptides C7, C12, and CLP-2 in DU145 cells at various concentrations. Triton X-100 (0.5% v/v) is used as positive control. Percentage of remaining peptides CLP-2 (B), C7 (C), and C12 (D) in human serum. Data are presented as mean ± SD (n = 3).

Figure 7.. Anti-tumor activity of the anti-PD-L1 peptides.

(A) Balb/c mice bearing CT-26 tumor cells received anti-PD-L1 peptides or antibody (0.5 mg/kg) by intraperitoneal injection daily. (B) Measurement of tumor growth over time. (C) Tumor growth inhibition (TGI) percentage on day 18. (D) Survival curves. Tumor progression curves of individual mice (red-female, black-male) in (E) saline, (F) CLP-2, (G) C12, (H) anti-PD-L1 antibody, and (I) C7 groups, respectively. ( ** p < 0.01)