The human acute T lymphocytic leukemia cell line (Jurkat T cells) and the human tongue squamous cell carcinoma cell line (Cal27 cells) were purchased from the cell bank of the typical Culture Preservation Committee of the Chinese Academy of Science (Shanghai, China). The Jurkat cells were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (04-001-1ACS, Biological Industries, Kibbutz Beit-Haemek, Israel). The Cal27 cells were grown in Dulbecco’s modified Eagle’s medium/F12 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (10099-141, Gibco, Grand Island, NY). PBMCs were isolated from peripheral blood donated by healthy adults through density gradient centrifugation and cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (10099-141, Gibco), interferon γ (IFN-γ; 300-02, 2000 U/mL, PeproTech, Rocky Hill, NJ), and IL-2 (200-02, 10 ng/mL, PeproTech).

We searched for the key regions through which human PD-L1 binds to human PD-1 and compared the amino acid sequences in the key regions of PD-L1 with the sequence of nABP284, which was identified by phage display technology in our previous study. The ²RLKEIA⁷ motif in nABP284 (SRLKEIANSPTQFWRMVARNTLGNGAKQSLNIEHARL) was considered the key region for specific binding. Based on the cleft between Y68 and E136 of PD-1 near the key binding region, which was just enough to accommodate the circular structure on the amino acid side chains, we hypothesized that adding histidine residues could enhance the binding affinity of nABP284 for PD-1 considering that histidine has an imidazole ring; this hypothesis was confirmed by AutoDock and PyMOL simulations. Thus, we added branched peptide (SHHHRL) to the N-terminus of nABP284 to obtain nABPD1. The new peptide was synthesized by China Peptide Company (Jiangsu, China) through solid-phase synthesis, and electrospray ionization–mass spectrometry/high-performance liquid chromatography (ESI-MS/HPLC) confirmed the sequence of the new peptide and its purity.

We used the Biacore T100 platform (GE Healthcare, Pittsburgh, PA) to measure the binding affinity of peptides through surface plasmon resonance (SPR). The surface of the chip (CM5) was esterified using the crosslinking agent EDC/NHS at pH 4.5, human recombinant PD-1 protein (PD1-H5221, Acrobiosystems, Newark, NJ) was conjugated to the chip (CM5) at a concentration of 5 μg/mL in coupling buffer (10 mM sodium acetate, pH 4.5), and excess active carboxyl groups on the surface of the CM5 chip were blocked with ethanol hydrochloride (pH 8.5). The channel that did not bind the human recombinant PD-1 protein was set as the reference. All the SPR signals were calibrated by subtraction of the reference channel response. The peptides were diluted in running buffer (pH 7.4, 100 mM Tris, 150 nM NaCl, 0.005% Tween-20). We first conducted the experiment with 1.25 μM (nABP284) and 0.125 μM (nABPD1), and we chose the concentration gradient of peptides according to the response values. For nABP284 analysis, seven concentrations (20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 μM) were used for a round of SPR measurements; similarly, for nABPD1, seven concentrations (2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μM) were used for a round of SPR measurements. The CM5 chip was regenerated with 10 mM glycine-HCl (pH 2.5) after each round of association and dissociation. The affinity (K_D value) was calculated by fitting the peptide binding curve on the blank subtracted sensorgrams (Biacore Evaluation Software, Cytiva, Marlborough, MA).

In the following experiments, an experimental group and a control group were included. For the experimental group, phorbol 12-myristate 13-acetate (PMA; 1652981, 50 ng/mL, PeproTech) and ionomycin calcium salt (ionomycin, 5608212, 1 μg/mL, PeproTech) were used to stimulate the expression of PD-1 in Jurkat T cells for 24 hours, while unstimulated Jurkat T cells expressing low levels of PD-1 served as the control group. For immunofluorescence analysis, Jurkat T cells (1×10⁶) were harvested and blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich) at room temperature (RT) for 1 hour to reduce nonspecific binding. After blocking, the cells were collected and incubated with 10 μM fluorescein isothiocyanate (FITC)–conjugated PD-1–binding peptides at 37°C for 1 hour. Then, the cells were washed with phosphate buffered saline (PBS) three times and fixed with 4% paraformaldehyde. After three washes with PBS, the cells were incubated with an anti–PD-1 antibody (ab52587, 1:100, Abcam, Cambridge, MA) overnight at 4°C. After three washes with PBS, a goat polyclonal secondary antibody against mouse IgG (ab150115, 1:500, Abcam) was allowed to bind to the anti–PD-1 antibody for 1 hour at 4°C. After three washes with PBS, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1,000, Sigma-Aldrich), and the cells were mounted with fluorescence mounting medium (S3023, DAKO, Carpinteria, CA). Furthermore, a competition assay was performed using confocal microscopy, and the images were analyzed by ImageJ (National Institutes of Health, Bethesda, MD). For flow cytometry analysis, cells (2×10⁵) were harvested and blocked with 2% BSA (Sigma-Aldrich) for 1 hour at RT and then incubated with 10 or 40 μM FITC-conjugated PD-1–binding peptides for 1 hour at 4°C. An anti–PD-1 antibody was used as a positive control. After blocking, the cells were incubated with a PD-1 antibody (130-120-389, 1:50, Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 minutes at 4°C. After three washes with PBS, the cells were resuspended in PBS and analyzed with a flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA).

Jurkat T cells were divided into two groups. For the experimental group, cells were pre-stimulated with PMA (1652981, 50 ng/mL, PeproTech) and ionomycin calcium salt (ionomycin, 5608212, 1 μg/mL, PeproTech) for 24 hours, and unstimulated Jurkat T cells were used as the control group. The cells (5×10⁵) were harvested and washed once with fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% BSA). Biotinylated human PD-L1 (PD1-H82F3, Acro Biosystems) was diluted with FACS buffer to a concentration of 4 μg/mL. PD-1–binding peptides were diluted to various concentrations (0.8, 2.66, 8, 26.6, and 80 μM) with FACS buffer. Then, two working solutions (PD-L1 protein diluent and peptide diluent) were mixed well in equal volumes, and 100 μL of each mixture was added to the tube with the cell pellet and incubated at 4°C for 1 hour. The cells were washed with FACS buffer three times, and APC streptavidin diluent (405207, 0.12 μg/mL, BioLegend, San Diego, CA) was incubated with the cells at 4°C for 1 hour. The cells were washed with FACS buffer three times, resuspended in 500 μL of PBS, transferred to a flow tube and analyzed by flow cytometry (CytoFLEX, Beckman Coulter).

Cytotoxicity assays were performed using a Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Jurkat T cells (4,000 cells per well), ICIK cells (4,000 cells per well), or Cal27 cells (2,000 cells per well) were seeded in 96-well plates and cultured for 24 hours. Various concentrations of each peptide (1, 2, 4, 8, 16, 32, and 64 μM) were added to the culture medium. The cells were cultured in a cell incubator for 24 hours, after which 10 μL of CCK-8 solution was added to each well, and the mixture was cultured in a cell incubator for another 2 hours. A Victor X5 Multilabel Plate Reader (PerkinElmer, Singapore) was used to measure the absorbances of each well at 450 nM. Cell viability (%) was calculated using the following formula: cell viability (%)=[(A_sample–A_baseline)/(A_control–A_blank)]×100%. A_sample represents the absorbance of the experimental well (containing cells, CCK-8 solution, medium, and peptide diluent); A_baseline represents the absorbance of the baseline well (containing no cells, CCK-8 solution, medium, and peptide diluent); A_control represents the absorbance of the control well (containing cells, CCK-8 solution, medium, and no peptide diluent); and A_blank represents the absorbance of the blank well (containing no cells, CCK-8 solution, medium, and no peptide diluent).

Jurkat T cells were stimulated with PMA (1652981, 50 ng/ mL, PeproTech) and ionomycin calcium salt (ionomycin, 5608212, 1 μg/mL, PeproTech) for 24 hours. Cal27 cells were stimulated with 500 U/mL IFN-γ (300-02, PeproTech) for 48 hours. Stimulated Jurkat T cells were pre-incubated with nABP284, nABPD1 (10 μM) or a functional-grade PD-1 monoclonal antibody (16-9989-82, 2 μg/mL, J116, eBioscience, Thermo Fisher Scientific, Waltham, MA) for 1 hour at 37°C. Subsequently, 1×10⁴ Cal27 cells were seeded in 96-well plates, and after the Cal27 cells had adhered, the supernatant was discarded, and Jurkat T cells were added to the wells at a ratio of 4:1 with Cal27 cells in 200 μL of medium. The supernatant was collected after 24 hours of coculture, and the IL-2 levels were measured with an IL-2 Human Uncoated ELISA (enzyme-linked immunosorbent assay) kit (88-7025-86, Thermo Fisher Scientific) according to the manufacturer’s instructions. Finally, the absorbance was determined with a Victor X5 Multilabel Plate Reader (PerkinElmer) at 450 nM.

After mixing human peripheral blood with PBS at a ratio of 1:1, the mixture was added on top of the same volume of Ficoll solution (17-1140-02, GE Healthcare). Density gradient centrifugation (400 ×g, 20 minutes) was performed to separate the components of the human peripheral blood. After isolation, PBMCs were added to Petri dishes coated with 10 μg/mL GMP-grade anti-CD3 mAb (clone OKT3, T210, Takara, Tokyo, Japan) and incubated at 4ºC overnight; then, the cells were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (10099-141, Gibco), IFN-γ (300-02, 2000 U/mL, PeproTech), and IL-2 (200-02, 10 ng/mL, PeproTech). The medium was changed every 3 days, and the cells were cultured for 10 days. We called these activated PBMCs ICIK cells. To analyze ICIK cell-mediated killing, Cal27 cells were stimulated with 500 U/mL IFN-γ (300-02, PeproTech) for 48 hours, and ICIK cells were pre-incubated with nABP284, nABPD1 (10 μM), or a functional-grade PD-1 monoclonal antibody (16-9989-82, 2 μg/mL, J116, eBioscience, Thermo Fisher Scientific) for 1 hour at 37°C. Subsequently, 1×10⁴ Cal27 cells were seeded in 96-well plates. After the Cal27 cells had adhered, the supernatant was discarded, and ICIK cells were added to the Cal27 cells in each well at a ratio of 10:1 in 100 μL of medium. The 96-well plates were centrifuged at 250 ×g for 4 minutes to ensure that the ICIK cells and Cal27 cells came into contact with each other. After 6 hours of incubation, lactate dehydrogenase (LDH) levels in the medium were measured with a CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (G1780, Promega, Madison, WI) according to the manufacturer’s instructions, and the absorbance was measured with a Victor X5 Multilabel Plate Reader (PerkinElmer) at 490 nM.

Statistical analyses were performed by GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA). All the data were analyzed by Student’s t test or one-way ANOVA. The data are the mean±standard error of three separate experiments.

We determined that the K_D value of nABPD1 was 11.9 nM (Fig. 2B), which was significantly higher than that of nABP284 (Fig. 2A). Unstimulated Jurkat T cells expressed very low levels of PD-1; conversely, PMA-stimulated Jurkat T cells showed high PD-1 expression (Fig. 3A). To demonstrate the binding specificity of nABPD1 to PD-1, we allowed nABPD1 to bind to Jurkat T cells before and after stimulation. As exhibited in Fig. 3A, nABPD1 could bind to Jurkat T cells in which PD-1 expression was stimulated by PMA and ionomycin, while very little nABPD1 bound to unstimulated Jurkat T cells. The new peptide had a higher rate of binding to PD-1–expressing Jurkat T cells than nABP284, and this increase in the binding rate was dose-dependent (Fig. 3B and C). These results indicate that the binding of nABPD1 to PD-1–expressing Jurkat T cells is specifically mediated by PD-1.

The purpose of these studies was to determine whether the enhanced binding ability of the new peptide compared with that of nABP284 resulted in enhanced blocking and neutralizing capacity. Jurkat T cells were pre-stimulated with PMA and ionomycin for 24 hours. After 1 hour of incubation with nABP284, nABPD1, or a functional anti–PD-1 antibody, the amount of PD-1 that was bound on the surface was reduced in treated Jurkat T cells compared with untreated Jurkat T cells (Fig. 4A). Quantification of the mean fluorescence intensity indicated that both nABP284 and nABPD1 had the ability to block PD-1 on the surface of stimulated Jurkat T cells and that nABPD1 had a better blocking capacity than nABP284 at the same concentration (Fig. 4B).

Next, we examined the ability of nABP284 and nABPD1 to neutralize recombinant human PD-L1 protein. As the ligand for PD-1, PD-L1 can specifically bind to PD-1. Before allowing it to bind stimulated Jurkat T cells expressing PD-1, we mixed nABP284 or nABPD1 with recombinant human PD-L1 protein at various concentrations. The binding rate of the recombinant human PD-L1 protein was determined by flow cytometry, and at concentrations of 4, 13.3, and 40 μM, the neutralization capacities of nABPD1 and nABP284 were significantly different (Fig. 5A); additionally, the binding of human PD-L1 recombinant protein to stimulated Jurkat T cells overexpressing PD-1 was inhibited by increasing the concentration of nABP284 or nABPD1. The half maximal inhibitory concentrations (IC₅₀) of nABP284 and nABPD1 were 9.610 and 2.585 μM, respectively (Fig. 5B). The neutralization capacity of nABPD1 was significantly enhanced compared with that of nABP284 (Fig. 5A and B).

After stimulation of Jurkat T cells with PMA and ionomycin, PD-1 was highly expressed on the surface of the cells (Fig. 3A). Furthermore, Jurkat T cells secreted a large amount of IL-2. Similarly, the expression of PD-L1 on the surface of Cal27 cells increased after pre-incubation with increasing IFN-γ concentrations (Fig. 6A). The cytokine secretion level is an important indicator of T cell function. After coculture, Cal27 cells inhibited Jurkat T cells through the interaction of PD-1 with PD-L1, which was manifested by a decrease in IL-2 levels in the medium, as determined by ELISA (Fig. 6B). To examine whether the new peptide is more effective than nABP284 in blocking the interaction between PD-1 and PD-L1, we first determined that nABP284 and nABPD1 were not toxic to Cal27 cells and Jurkat T cells (Fig. 6C), and then, we added nABP284, nABPD1, or a functional anti–PD-1 antibody to the coculture system. The results showed that the IL-2 level was significantly increased in the coculture systems to which nABP284, nABPD1, or the anti–PD-1 anti-body was added compared to the control coculture system (Fig. 6B). Hence, nABP284 and the new peptide rescued the inhibitory effect on Jurkat T cells by blocking the interaction of PD-1 and PD-L1, and the blocking effect of nABPD1 was significantly better than that of nABP284.

As shown in Fig. 6D, PBMCs were isolated from peripheral blood drawn from volunteers and activated by stimulation with a series of cytokines, including OKT-3, IL-2, and IFN-γ. We called these activated PBMCs ICIK cells. Flow cytometry showed that ICIK cells significantly overexpressed PD-1 compared with freshly isolated PBMCs (Fig. 6D). ICIK cells were cocultured with IFN-γ–stimulated Cal27 cells, and the cell-mediated cytotoxicity of ICIK cells to Cal27 cells was evaluated by measuring the concentration of LDH in the medium by ELISA. Due to the PD-1/PD-L1 pathway, the cell-mediated cytotoxicity of ICIK cells to Cal27 cells was relatively weak (Fig. 6E). The cytotoxicity of ICIK cells to Cal27 cells was significantly increased in the nABPD1-treated group compared with the control group and nABP284 group (Fig. 6E). Considering that nABPD1 has no direct ability to kill Cal27 cells (Fig. 6C), the above experimental results suggest that nABPD1 can enhance ICIK cell-mediated cytotoxicity by blocking the interaction between PD-1 and PD-L1 (Fig. 6D and E).