Site-Selective Functionalized PD-1 Mutant for a Modular Immunological Activity against Cancer Cells

Targeting immune checkpoints is a well-established strategy in cancer therapy, and antibodies blocking PD-1/PD-L1 interactions to restore the immunological activity against cancer cells have been clinically validated. High-affinity mutants of the PD-1 ectodomain have recently been proposed as an alternative to antibodies to target PD-L1 on cancer cells, shedding new light on this research area. In this dynamic scenario, the PD-1 mutant, here reported, largely expands the chemical space of nonantibody and nonsmall-molecule inhibitor therapeutics that can be used to target cancer cells overexpressing PD-L1 receptors. The polyethylene glycol moieties and the immune response-stimulating carbohydrates, used as site-selective tags, represent the proof of concept for future applications.


Cell culture
The MDA-MB-231 is a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer (TNBC) cell line as it lacks estrogen receptor (ER), and progesterone receptor (PR) expression, as well as human epidermal growth factor receptor 2 (HER2).MDA-MB-231 cells were cultivated in DMEM medium complete with inactivated 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% L-glutamine.MCF-7 represents an ER-positive breast cancer model expressing both ERα and Erβ, PR as well as HER2 receptors.MCF-7 cells were cultivated in EMEM medium supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate and 1% insulin.Both cell cultures were kept in an incubator at 37°C in a humidified atmosphere with a CO 2 pressure of 5%.Human Peripheral Blood Mononuclear Cells (PBMC), were isolated from the buffy coat of healthy volunteers after their informed consent.PBMC were separated from whole blood by density gradient (ficoll) centrifugation.Isolated PBMC were cultivated in RPMI medium, completed with 10% FBS, 1% Kanamycin solution, 1% L-glutamine, 1% non-essential amino acids and 1% sodium pyruvate.To evaluate the expression of PD-L1 on cell lines, MDA MB 231 and MCF-7 cells were harvested labelled with PE-conjugated anti-PD-L1 antibody and the level of PD-L1 expression analyzed by flow cytometry.

Cell co-culture
For co-culture experiments, each cell line was plated in a 96 well tissue culture-treated plate at a concentration of 30000 cells/well.Cells were kept in a humidified incubator at 37°C in 5% CO 2 for 24 h to allow the cell to adhere.PBMC were activated or not activated by PHA 10 μg/mL overnight treatment.To establish co-culture cell lines were treated with 10 μg/mL or 50 μg/mL of WT PD-1, HACTR-PD-1, 1-or 2-HACTR-PD-1-L-rhamnose for 1 hour, followed by the addition of inactivated/activated PBMCs (3 x 10^5 cells/well).To distinguish the effects of co-culturing PBMCs with breast cancer cells from PBMC activation, two controls were used; activated PBMCs cultured alone (negative control) or co-cultured with breast cancer cells from each cell line, in the absence of recombinant or natural proteins.

Cytotoxicity assay
The effect of WT PD-1, HACTR-PD-1 or HACTR-PD-1-L-rhamnose on T cell cytotoxic activity was analyzed using Calcein-AM cytotoxicity assay.MDA-MB-231 and MCF-7 cells were labelled with 1 mM CAM at 37°C for 15 min, washed, and seeded in a 96-well plate at a density of 3 x 10 4 cells in 50 μL per well.The following day, labelled target cells were treated with 10 μg/mL or 50 μg/mL of WT PD-1, HACTR-PD-1, 1-or 2-HACTR-PD-1 for 1 hour and incubated at a 1:10 ratio with overnight stimulated PBMC (PBMC stimulation was performed as described in paragraph Cell Co-Culture).Each plate included target cells alone, as controls, for spontaneous cell death measurements.Plates were incubated at 37°C in a humidified atmosphere with 5% CO 2 for 24h.After incubation, the cells of each well were harvested, washed and labelled with propidium iodide (PI), and the cytotoxicity was measured by flow cytometry (FACS).Live target cells were identified as CAM high /PI -population, whereas killed target cells were CAM low /PI + and the effector cells were CAM -(at least 10-fold less fluorescent than killed target cells).After gating on target cells, cytotoxicity was calculated as the % increase in CAM low /PI + population relative to target cells alone [cytotoxicity, % = (CAM low /PI + in experimental wells -CAM low /PI + in control wells)/CAM high /PI -in control well x 100].The mean cytotoxicity % SEM for each condition was calculated from three replicate experimental wells.

Flow cytometry
Flow cytometry analyzes were performed using the Accuri C6 (Thermo Fisher Scientific, Italy).Forward (FCS) and side (SSC) scatters were used to identify cell populations and measure size and granularity of the cells.Auto-fluorescence was recorded by analyzing unstained cells in the FL-1 channel (blue laser; excitation 488, emission 530/30).For detection of cell surface markers 1 mg/mL of monoclonal mouse anti-human antibodies CD14-FITC, CD86-PE, CD11b-PeCy7 were used for each sample.THP1 cells (2 x 10 5 cells/mL) were seeded in 6 MW and differentiated into macrophages as described.Differentiate macrophages (M0) were treated with increasing concentrations (0.1 -10 mg/mL) of tested compounds or with 0.5 mg/mL (LPS).After 24h incubation, cells were harvested, washed, incubated with the antibodies for 30 minutes on ice in the dark.Labelled samples were washed, resuspended in PBS and analyzed by FACS, for each sample 10000 events were recorded.All data was analyzed using FCS express 7 (Flow cytometry software, DeNovo software).

ELISA assay
To evaluate the effect of new PD-1 mutants on T helper/reg cell activity co-culture experiments were performed as described previously.After 48h of co-culture, plates were centrifuged at 1500 rpm for 5 minutes and cell supernatants collected and stored at -80°C until the analysis.IFN-γ and IL-10 quantification in the culture media was performed by ELISA, following the manufacturer's instructions.Absorbance at 450 nm was monitored with a microplate reader.THP1 cells (2 x 10 5 cells/mL) were seeded in 6 MW and differentiated into macrophages as described.Differentiate macrophages (M0) were treated with increasing concentrations (0.1 -10 mg/mL) of tested compounds or with 0.5 mg/mL (LPS).After 24h incubation culture media were collected and stored in -80°C until analysis.Levels of IL-8, TNF-a and IL-10 were measured by ELISA assay according to manufacturer's guidelines (Biolegend® San Diego, CA, USA).

Cell subset characterization
To evaluate the effect of WT PD-1, HACTR-PD-1, 1-or 2-HACTR-PD-1 on T cell subset frequency co-culture experiments were performed as described previously.After 72 h of co-culture PBMC were harvested labelled for 30 min on ice with anti-human CD3, CD4, CD8 and CD25 and washed 2 times with PBS.CD3, CD4, CD8, and CD25 frequency were analyzed by FACS.

Statistical analysis
Results are expressed as means ± SEM of at least three independent experiments.Independent experiments were conducted using PBMC from at least 3 different donors.Statistical significance was evaluated by the one-way ANOVA followed by the Student's t test for unpaired populations, using Graph Pad Prism 9 (Graph Pad Software, Inc., San Diego, CA, USA).Differences were considered statistically significant when p < 0.05.

NMR Titrations of the functionalized HACTR-PD-1 mutant with PD-L1
The interactions of the free and functionalized HACTR-PD-1 mutant with PD-L1 have been investigated through solution NMR titrations.During the NMR titration, increasing aliquot of PD-L1 [to reach the concentrations of 12.5, 25, 50 µmol•dm -3

Figure S2
. 2D 1 H-15 N HSQC overlaid spectra of free HACTR-PD-1 (black) with respect to HACTR-PD-1 in the presence of PD-L1 (in 1:1 molar ratio, red).The spectra were acquired on a spectrometer operating at 900 MHz, 1 H Larmor frequency, and 298 K.The spectrum of the complex was acquired with a higher number of scans than the reference spectrum.

Figure S4
. 2D 1 H-15 N HSQC overlaid spectra of HACTR-PD-1 conjugated with PEG 5 kDa (black) and HACTR-PD-1 conjugated with PEG 5 kDa in the presence of PD-L1 (in 1:1 molar ratio, red).The spectra were acquired on a spectrometer operating at 950 MHz, 1 H Larmor frequency, and 298 K.The spectrum of the complex was acquired with a higher number of scans than the reference spectrum.The spectra were acquired on a spectrometer operating at 700 MHz, 1 H Larmor frequency, and 298 K.The spectrum of the complex was acquired with a higher number of scans than the reference spectrum.

Figure S1 .
Figure S1.Secondary structure prediction obtained by the program TALOS+ using the experimental values of chemical shifts of HN, N, C', Cα, and Cβ atoms as input data.The blue bars indicate the β-strand propensity.

Figure S6 .
Figure S6.2D 1 H-15 N HSQC overlaid spectra of HACTR-PD-1 conjugated with L-rhamnose, 1-HACTR-PD-1 (black) and HACTR-PD-1 conjugated with L-rhamnose in the presence of PD-L1 (in 1:1 molar ratio, red).The spectra were acquired on a spectrometer operating at 700 MHz, 1 H Larmor frequency, and 298 K.The spectrum of the complex was acquired with a higher number of scans than the reference spectrum.

Figure S7 .
Figure S7.PD-L1 level expression on MDA MB 231 and MCF-7 breast cancer cell lines.Representative overlapping histogram plots of PD-L1 level of expression on MDA MB 231 and MCF-7 cells analyzed by FACS; isotype control black; PD-L1 l blue.Data represent one of at least three independent experiments.

Figure S8 .
Figure S8.Effects of test compounds on THP1 cells morphological changes.(A) Representative forward light scatter and side light scatter plots of THP1 cells treated 24 h with 150 nM of PMA and of M0 treated (24 h) with 0.5 g/mL of LPS or 1 g/mL of tested compounds.(B) Representative histograms of autofluorescence of THP1 cells treated 24h with 150 nM of PMA and of M0 treated (24 h) with 0.5 g/mL of LPS or 1 g/mL of tested compounds.Data are representative of at least three experiments.

Figure S10 .
Figure S10.Effects of tested compound on cytokines secretion in differentiated THP1 cells.PMA differentiated THP1 cells were treated with 0.5 g/mL of LPS or 1 g/mL of tested compounds for 48h, cells culture medium harvested and IL-8 (A) and TNF- (B) levels measured by ELISA assay.Results represent mean ± SEM of at least three independent experiments.* 0.05 treated vs. control (CTRL).
The crude was purified via flash chromatography on silica gel (CH 2 Cl 2 :CH 3 OH / 95:5), to yield compound 9 as a pale yellow oil (140 mg, 50 % yield over two steps).

Synthesis of compound 10:
To a solution of 16 (249 mg, 0.73 mmol) in ethanol (10 mL), Pd/C (10 % wt, 189 mg) was added under a N 2 atmosphere.The reaction was stirred at room temperature, under H 2 atmosphere.After 16 h the suspension was filtered through a pad of Celite ® , the solvent was removed under vacuum to give pure 17 (150 mg, quant.yield) as a cerous solid.