Cancer Res Treat.  2022 Apr;54(2):362-374. 10.4143/crt.2021.424.

Engineering a High-Affinity PD-1 Peptide for Optimized Immune Cell-Mediated Tumor Therapy

• Chen Y
• Huang H
• Liu Y
• Wang Z
• Wang L
• Wang Q
• Zhang Y
• Wang H

• KMID: 2528206
• DOI: http://doi.org/10.4143/crt.2021.424

Abstract

Purpose
The purpose of this study was to optimize a peptide (nABP284) that binds to programmed cell death protein 1 (PD-1) by a computer-based protocol in order to increase its affinity. Then, this study aimed to determine the inhibitory effects of this peptide on cancer immune escape by coculturing improving cytokine-induced killer (ICIK) cells with cancer cells.
Materials and Methods
nABP284 that binds to PD-1 was identified by phage display technology in our previous study. AutoDock and PyMOL were used to optimize the sequence of nABP284 to design a new peptide (nABPD1). Immunofluorescence was used to demonstrate that the peptides bound to PD-1. Surface plasmon resonance was used to measure the binding affinity of the peptides. The blocking effect of the peptides on PD-1 was evaluated by a neutralization experiment with human recombinant programmed death-ligand 1 (PD-L1) protein. The inhibition of activated lymphocytes by cancer cells was simulated by coculturing of human acute T lymphocytic leukemia cells (Jurkat T cells) with human tongue squamous cell carcinoma cells (Cal27 cells). The anticancer activities were determined by coculturing ICIK cells with Cal27 cells in vitro.
Results
A high-affinity peptide (nABPD1, K_D=11.9 nM) for PD-1 was obtained by optimizing the nABP284 peptide (K_D=11.8 μM). nABPD1 showed better efficacy than nABP284 in terms of increasing the secretion of interkeulin-2 by Jurkat T cells and enhancing the in vitro antitumor activity of ICIK cells.
Conclusion
nABPD1 possesses higher affinity for PD-1 than nABP284, which significantly enhances its ability to block the PD-1/PD-L1 interaction and to increase ICIK cell-mediated antitumor activity by armoring ICIK cells.

Keyword

PD-1; Peptide optimization; Affinity; Computer simulation; Immune checkpoint inhibitor; Improving cytokine-induced killer (ICIK) cells; Immunotherapy

Figure

• 

  Fig. 1 Optimization and improvement of the peptide. (A) Main binding regions between human programmed cell death protein 1 (hPD-1) and human programmed death-ligand 1 (hPD-L1). The key amino acid residues through which it binds programmed cell death protein 1 (PD-1) are labeled. The region including A121 K124 R125 in PD-L1 shows sequence homology with nABP284. (B) AutoDock simulation provided evidence for the hypothesis that the imidazole ring on histidine could bind to the cleft between Y68 and E136 in PD-1. (C) Sequence of nABP284 and nABPD1, nABPD1 was optimized from nABP284 by adding branched chains (SHHHRL) with three consecutive histidine residues (HHH) on the N-termini. (D) Structural formula of HHH.

• 

  Fig. 2 Binding affinity of the peptides. The binding affinity of nABP284 (A) and nABPD1 (B) to human programmed cell death protein 1 (PD-1) recombinant protein was analyzed by surface plasmon resonance, and the affinity of nABPD1 for PD-1 was significantly enhanced compared with that of nABP284.

• 

  Fig. 3 Binding of the peptides to stimulated Jurkat T cells. (A) Expression of programmed cell death protein 1 (PD-1) and binding of 10 μM nABP284 or nABPD1 labeled with FITC (green) on Jurkat T cells before and after treatment with PMA (phorbol 12-myristate 13-acetate; 50 ng/mL) and ionomycin (1 μg/mL) for 24 hours. After allowing the peptide to bind, the cells were incubated with an anti–PD-1 antibody (red), and nuclei were stained with DAPI (blue). Images were merged. (B, C) Flow cytometry analysis of the binding of nABP284 or nABPD1 labeled with FITC to Jurkat T cells before and after stimulation. The data are presented as the mean±standard error of three independent experiments and were analyzed by Student’s t test. ***p < 0.001.

• 

  Fig. 4 Competition of peptides for binding to programmed cell death protein 1 (PD-1). (A) Images of the binding of nABP284, nABPD1 and an anti–PD-1 antibody to PD-1 expressed on the surface of Jurkat cells stimulated with PMA (phorbol 12-myristate 13-acetate) and ionomycin for 24 hours were obtained by confocal microscopy. Scale bars=12 μm. (B) Image J was used to measure the mean fluorescence intensity to evaluate PD-1 binding on the surface of stimulated Jurkat cells that were blocked with nABP284, nABPD1, or a functional PD-1 antibody in advance. The data are presented as the mean±standard error of three independent experiments and were analyzed by one-way ANOVA. ***p < 0.001; ns, not significantly different.

• 

  Fig. 5 Neutralization of programmed death-ligand 1 (PD-L1) with the peptides. (A) The neutralization capacities of nABP284 and nABPD1 at concentrations of 4, 13.3, or 40 μM. (B) Flow cytometry showed that the binding of human PD-L1 recombinant protein to stimulated Jurkat cells overexpressing programmed cell death protein 1 was inhibited by increasing the concentrations of nABP284 or nABPD1. nABPD1 showed a stronger blocking effect than nABP284 at the same concentration. The IC50 values of nABP284 and nABPD1 were 9.610 and 2.585 μM, respectively. The concentration of PD-L1 was 2 μg/mL. The data are presented as the mean±standard error of three independent experiments and were analyzed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significantly different.

• 

  Fig. 6 In vitro analysis of peptide function. (A) Expression of programmed death-ligand 1 (PD-L1) on cal27 cells after 48 hours of stimulation with different concentrations of interferon γ (IFN-γ). (B) Coculture with Cal27 cells significantly reduced the amount of interleukin 2 (IL-2) secreted by Jurkat T cells, and this inhibitory effect was reversed by nABP284 or nABPD1. nABPD1 showed a better effect than nABP284. (C) The Cell Counting Assay (Cell Counting Kit-8) revealed that nABP284 and nABPD1 were not directly cytotoxic to stimulated Cal27 cells, Jurkat T cells, or improving cytokine-induced killer (ICIK) cells at a concentration of 64 μM after 24 hours of culture. (D) Venous blood was drawn from donators, and peripheral blood monocytes (PBMCs) were isolated from the venous blood by density gradient centrifugation with Ficoll. OKT3, IL-2, and IFN-γ were used as shown in the image above to stimulate PBMCs. These stimulated PBMCs were called ICIK cells. The expression of programmed death 1 (PD-1) on PBMCs and ICIK cells was analyzed by flow cytometry. (E) Compared with untreated group and nABP284 group, nABPD1 significantly enhanced the cytotoxicity of ICIK cells to Cal27 cells. The data are presented as the mean±standard error of three independent experiments and were analyzed by one-way ANOVA. *p < 0.05, ***p < 0.001; ns, not significantly different.

Reference

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