PD-L1 targeted peptide demonstrates potent antitumor and immunomodulatory activity in cancer immunotherapy

Abstract

Background: In recent years, immunotherapy has been emerging as a promising alternative therapeutic method for cancer patients, offering potential benefits. The expression of PD-L1 by tumors can inhibit the T-cell response to the tumor and allow the tumor to evade immune surveillance. To address this issue, cancer immunotherapy has shown promise in disrupting the interaction between PD-L1 and its ligand PD-1.

Methods: We used mirror-image phage display technology in our experiment to screen and determine PD-L1 specific affinity peptides (PPL-C). Using CT26 cells, we established a transplanted mouse tumor model to evaluate the inhibitory effects of PPL-C on tumor growth in vivo. We also demonstrated that PPL-C inhibited the differentiation of T regulatory cells (Tregs) and regulated the production of cytokines.

Results: In vitro, PPL-C has a strong affinity for PD-L1, with a binding rate of 0.75 μM. An activation assay using T cells and mixed lymphocytes demonstrated that PPL-C inhibits the interaction between PD-1 and PD-L1. PPL-C or an anti-PD-L1 antibody significantly reduced the rate of tumor mass development in mice compared to those given a control peptide (78% versus 77%, respectively). The results of this study demonstrate that PPL-C prevents or retards tumor growth. Further, immunotherapy with PPL-C enhances lymphocyte cytotoxicity and promotes proliferation in CT26-bearing mice.

Conclusion: PPL-C exhibited antitumor and immunoregulatory properties in the colon cancer. Therefore, PPL-C peptides of low molecular weight could serve as effective cancer immunotherapy.

Keywords: PD-L1 binding peptide; PPL-C; colon cancer; immune checkpoint inhibitors; immunotherapy.

Figure 1

(A) Mechanisms of how PD-L1–binding peptides activate PD-L1–mediated inhibition of T cells. (B) Schematic description of the PD-L1–binding peptides by phage display library screening procedure.

Figure 2

The physicochemical properties of PPL-C peptides. PPL-C binding specificity to hPD-L1, mPD-L1, and BSA was analyzed by fluorescence-based ELISA (A, n = 3). Ligand inhibition assay analysis of the changes in fluorescence signals of PPL-C-FITC (green and blue lines) and PD-1-PE (red lines) with the increased concentration of peptide was analyzed by fluorescence-based ELISA (B, n = 3). The binding ability of PPL-C (blue lines) or PD-L1 mAb (red lines) to CHO (gray) and CHO-PD-L1 cells was determined by flow cytometry (C, n = 3). Statistical analysis of the results of three repeated flow experiments, where Pos% is the fluorescence positive rate, and Mean and Median are the mean fluorescence intensity values and median fluorescence intensity values (D, n = 3). PD-L1 expression on CT26 was detected by flow cytometry (E). Different concentrations of PPL-C/FITC peptides and a single concentration of biotinylated PD-1 were incubated simultaneously to detect PD-1 binding capacity on LLC, CT26 and MGC-803 cell lines (F). Data, mean ± SEM; *,P < 0.05; **,P < 0.01.

Figure 3

PPL-C reactivated T-cell function was verified by T-cell activation assays (A; n = 3) and MLR assay (B; n = 3). T-cell activation was positively correlated with IFN-γ production. Data, mean ± SEM; *,P < 0.05; **,P < 0.01.

Figure 4

PPL-C inhibited tumor growth in a transplantation tumor. Treatment schedule for mice bearing subcutaneous CT26 tumour of different tumor stages for the immunotherapy (A) and treatment schedule for mice growing to 20 mm³ for immunotherapy (C). Growth inhibition of early tumor pathology via PPL-C (B, D, n = 3). Survival prolonging curves for CT26-tumor-bearing Balb/c mice treated with various treatment methods (E, n = 6). Data, mean ± SEM; ***, P < 0.001.

Figure 5

FACS analysis of the spleen from a Balb/c mouse 14 days after challenge with various treatments. Flow cytometric analysis of the comparative in vivo effect of PPL-C and PD-L1 on regulatory cells (Tregs, CD4+CD25+Foxp3+) in CT26-bearing mice (A, B). The representative plots show the percentages of total splenic lymphocytes in CD3+CD8+ (cytotoxic T cell, C) and CD49+NKp46+ (NKT, D). Numbers in the corners indicate the percentage of cells in the gate. The data in (B) are shown as mean ± SEM, **, P < 0.01 (n = 3).

Figure 6

Representative images of CD8 and IFN-γ immunostaining on spleen tissues for each group (A). Representative images of CD8, IFN-γ and GZMA immunostaining on tumor tissues for each group (B). Scale bars, 50 μm. Magnification: 40×.

Figure 7

Lymphocyte cytotoxicity (A, n = 6) and proliferation (B, n = 6) analysis of spleen from a CT26-tumor-bearing mouse 14 days after challenge with various treatments. Data, mean ± SEM; *, P < 0.05; **, P < 0.01.

Figure 8

Splenocytes from various treatments were co-cultured with CT26 tumor cells in vitro for 48 and 72 hours. Supernatants were collected and IFN-γ was measured by an ELISA assay (A–F). ELISA assay analysis of serum from a CT26 tumor-bearing mouse 14 days after challenge with various treatments (G). This experiment was repeated twice with similar results. Data, mean ± SEM; *, P < 0.05; **, P < 0.01.