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. 2019 Oct 22;7(1):270.
doi: 10.1186/s40425-019-0705-y.

Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy

Hao Liu  1 , Zhen Zhao  1 , Li Zhang  1 , Yuanke Li  1 , Akshay Jain  1 , Ashutosh Barve  1 , Wei Jin  1 , Yanli Liu  1 , John Fetse  1 , Kun Cheng  2
Affiliations

Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy

Hao Liu et al. J Immunother Cancer. .

Abstract

Background: Immunotherapy using checkpoint inhibitors, especially PD-1/PD-L1 inhibitors, has now evolved into the most promising therapy for cancer patients. However, most of these inhibitors are monoclonal antibodies, and their large size may limit their tumor penetration, leading to suboptimal efficacy. As a result, there has been a growing interest in developing low-molecular-weight checkpoint inhibitors.

Methods: We developed a novel biopanning strategy to discover small peptide-based anti-PD-L1 inhibitors. The affinity and specificity of the peptides to PD-L1 were examined using various assays. Three-dimensional (3D) spheroid penetration study was performed to determine the tumor penetration capability of the peptides. Anti-tumor activity of the peptides was evaluated in mice bearing CT26 tumor cells.

Results: We discover several anti-PD-L1 peptide inhibitors to block PD-1/PD-L1 interaction. The peptides exhibit high affinity and specificity to human PD-L1 protein as well as PD-L1-overexpressing human cancer cells MDA-MB-231 and DU-145. Molecular docking studies indicate that the peptide CLP002 specifically binds to PD-L1 at the residues where PD-L1 interacts with PD-1. The peptide also blocks the CD80/PD-L1 interaction, which may further enhance the immune response of tumor-infiltrating T cells. Compared to antibody, the peptide CLP002 exhibits better tumor penetration in a 3D tumor spheroid model. The peptide CLP002 restores proliferation and prevents apoptosis of T cells that are co-cultured with cancer cells. The peptide CLP002 also inhibits tumor growth and increases survival of CT26 tumor-bearing mice.

Conclusions: This study demonstrated the feasibility of using phage display to discover small peptide-based checkpoint inhibitors. Our results also suggested that the anti-PD-L1 peptide represents a promising low-molecular-weight checkpoint inhibitor for cancer immunotherapy.

Keywords: CT26; Checkpoint inhibitor; PD-1; PD-L1; Peptide; Phage display; Tumor penetration.

PubMed Disclaimer

Conflict of interest statement

We are in the process of filing a patent for the anti-PD-L1 peptides discovered in this study.

Figures

Fig. 1
Fig. 1
Discovery of anti-PD-L1 peptides using a novel biopanning procedure. a The number of recovered phages from each round of biopanning. b Sequences of the discovered anti-PD-L1 peptides. c Binding affinities of the selected peptides towards human PD-L1 protein and albumin were measured using SPR. d Binding curves of the peptides on PD-L1-positive human cancer cells (MDA-MB-231 and DU-145) and PD-L1 deficient human cancer cells MCF-7. Binding curves of the anti-PD-L1 antibody were measured on DU-145 and MCF-7 cells. Results are represented as the mean ± SD (n = 3)
Fig. 2
Fig. 2
Blockade of the PD-1/PD-L1 interaction by the anti-PD-L1 peptides and antibody. a Blocking profile of the anti-human PD-L1 antibody (R&D, AF156) against human PD-L1 protein. b Blocking profile of the anti-human PD-L1 antibody (R&D, AF156) against DU-145 cells. c Blocking efficiency of the anti-PD-L1 peptides (10 μM) and the anti-human PD-L1 antibody (1 μM) against human PD-L1 protein. d IC50 and blocking efficiency of the peptides and antibody against human PD-L1 protein and human cancer cell line DU-145. e Blocking profiles of the peptides against human PD-L1 protein. f Blocking profiles of the peptides against DU-145 cells. g Blocking efficiency of the peptides and an anti-mouse PD-L1 antibody (BioXcell, 10F.9G2) at 10 μM against a mouse PD-L1 protein. h IC50 and blocking efficiency of the peptides against mouse PD-L1 protein and mouse cancer cell line 4 T1. i Blocking profiles of the peptides against mouse PD-L1 protein. j Blocking profiles of the peptides against mouse cancer cell line 4 T1. Results are represented as the mean ± SD (n = 3)
Fig. 3
Fig. 3
Molecular docking for the interaction between the anti-PD-L1 peptides and human PD-L1 protein (PDB ID: 5C3T). a Modeling of the interaction between CLP001 and PD-L1. b Modeling of the interaction between CLP002 and PD-L1. c Modeling of the interaction between CLP003 and PD-L1. d Modeling of the interaction between CLP004 and PD-L1. The PD-L1 residues responsible for peptide binding are highlighted in green. The binding residues for human PD-1 protein is highlighted in yellow. The overlapping PD-L1 residues for binding both anti-PD-L1 peptide and PD-1 protein are highlighted in pink
Fig. 4
Fig. 4
The CLP002 peptide restores T cell proliferation and prevents T cell apoptosis. Jurkat T cells were co-cultured with DU-145 cells and then incubated with the anti-PD-L1 peptides or antibody for 24 h. The CLP002 peptide and antibody restore Jurkat T cell proliferation (a) and reduces Jurkat T cell apoptosis (b-c) in the presence of PD-L1 overexpressing DU-145 cells. Results are represented as the mean ± SD (n = 3). (** p < 0.01; *** p < 0.001)
Fig. 5
Fig. 5
3D spheroid penetration of the CLP002 peptide and anti-PD-L1 antibody. 3D tumor spheroids of MDA-MB-231 cells were generated to compare the tumor penetration capability of the CLP002 peptide and the anti-PD-L1 antibody (BioXcell, 29E.2A3). Cy5-labeled peptide and antibody were incubated with the tumor spheroids (~ 700 μm in diameter) for 2 and 6 h, followed by confocal microscopy analysis to evaluate tumor penetration. a Representative Z-stacked confocal images of the spheroids with a z-step of 50 μm. The scale bar represents 200 μm. b The depth of penetration is quantified by mean fluorescence intensity. Results are represented as the mean ± SD (n = 3)
Fig. 6
Fig. 6
Anti-tumor activity of the anti-PD-L1 peptides and antibody. a CT26 tumor-bearing Balb/C mice (n = 10, 5 male and 5 female) were intraperitoneally injected with the anti-PD-L1 peptides (2 mg/Kg) daily for a total of 10 injections and the anti-mouse PD-L1 antibody (10 mg/Kg) every other day for a total of 5 injections. b Tumor volume measured over time. Tumor volume results were represented as the mean ± SE (n = 10). c Tumor growth curves of individual mice in each group. Image d and weight e of tumors harvested at day 14. The results were represented as the mean ± SD (n = 10). The expressions of IFNγ f, PD-L1 g and IL-6 h in harvested tumors were measured using ELISA. i The numbers of CD8+ T cells in each specimen were quantitated after immunohistochemical staining. Results were represented as the mean ± SD (n = 4). j Representative images of tumor specimen stained with anti-CD8 antibody. The scale bar represents 200 μm. (* p < 0.05; ** p < 0.01; *** p < 0.001)
Fig. 7
Fig. 7
Survival curves of the mice treated with the Anti-PD-L1 peptides and PD-L1 antibody. CT26 tumor-bearing Balb/C mice (n = 10, 5 male and 5 female) were intraperitoneally injected with the anti-PD-L1 peptides (2 mg/Kg) daily and the anti-mouse PD-L1 antibody (10 mg/Kg) every other day from day 4 to day 17. a Survival curves. GraphPad Prism 7 software (San Diego, CA) was used for statistical analysis. Comparison of two survival curves were conducted using the Gehan-Breslow-Wilcoxon test. b Tumor growth curves of individual mice in each group
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