Reagents and cell culture
FITC-labelled anti-His monoclonal antibodies (mAbs) were purchased from Abcam Company (Cambridge, UK). Mouse anti-His tag mAbs were purchased from Proteintech Group, Inc. (Wuhan, China). Goat anti-mouse IgG-HRP was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). APC-labelled rabbit anti-human PD-L1 mAbs were purchased from Sinobiological, Inc. (Beijing, China). A bispecific anti-human CD3/CD171 antibody was produced in-house. Atezolizumab was purchased from Selleck Chemical Company (Texas, USA). Recombinant human interleukin-2 (IL-2) was purchased from Huaxin Biotech Co., Ltd. (Shanghai, China). Human CXCL12 (hCXCL12, SDF-1α) was purchased from PeproTech (New Jersey, USA). The human interferon-γ (IFN-γ) ELISA kit was from R&D System (Minnesota, USA). Ficoll-Paque® PLUS was purchased from GE Healthcare (Wisconsin, USA).
Human peripheral blood mononuclear cells (hPBMCs) were provided by healthy volunteers (Changhai Hospital, Shanghai, China). Human cancer cell lines (Panc-1, AsPC-1, U251-MG and Jurkat cells) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Panc-1, AsPC-1 and Jurkat cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). U251-MG cells were cultured in DMEM supplemented with 10% FBS.
The anti-human PD-L1 VHH sequence used in this paper was previously reported , and the sequence of anti-human CXCR4 bivalent VHH was obtained from an issued patent (International Publication Number WO2011/161266A1). The two fragments were connected by a (G4S)3 linker to assemble the anti-PD-L1/CXCR4 bispecific nanobody (BsNb PX4). The sequence of the anti-PD-L1/CXCR4 nanobody with the C-terminal 6×His-tag was synthesized by GenScript® ProBIO (Nanjing, China) and cloned into the pET-22b (+) vectors. The pET-22b (+) vectors and targeted fragments were digested by Nco1 and EcoR1 restriction enzyme, with the ligation reaction using T4 DNA Ligase.
Expression and purification
The recombinant BsNb PX4 plasmid was transferred into Escherichia coli (E. coli) BL21 (DE3). Positive transformants were collected and cultured in Terrific Broth (TB) medium at 37 °C, and then the culture was induced with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG). After 18 h of induction at 25 °C, the cells were collected by centrifugation, resuspended in phosphate buffered saline (PBS), and lysed using a high-pressure homogenizer. The cell lysate was centrifuged at 12,000 r/min at 4 °C for 30 min to remove cell debris, and the supernatant was applied to a HisTrap affinity column (GE Healthcare, Wisconsin, USA). Then, the protein of interest was separated by step elution with 2 M imidazole, and the fractions containing the target protein were pooled and concentrated by using ultrafiltration tubes with a molecular weight cut-off (MWCO) of 10 kDa (Millipore, Tennessee, USA). The proteins were analysed by SDS-PAGE and transferred to a PVDF membrane. The blots were incubated with the mouse anti-His tag mAbs, followed by incubation with goat anti-mouse IgG-HRP. The protein bands were detected by an ECL Ultra Kit (NCM Biotech, Suzhou, China).
The purified bispecific nanobody (1 mg/mL) was concentrated in a 0.05 mmol/L NH4HCO3 solution. An ultrahigh pressure liquid system (ACQUITY I-Class) was connected to a VION IMS-Q-TOF (Waters, Massachusetts, USA). The MassPREP (Waters, Massachusetts, USA) desalting column was used for UPLC with a controlled column temperature of 60 ℃. Mobile phase A was a 0.1% formic acid-water solution, and mobile phase B was a 0.1% formic acid-acetonitrile solution. The system was preequilibrated with 95% mobile phase A, and then 0.5 µg of the bispecific nanobody was injected and eluted with a gradient at a flow rate of 0.2 mL/min. The mass spectrometry conditions were as follows: capillary voltage, 2.0 kV; sampling cone voltage, 60 V; ion source temperature, 115 °C; desolvent gas temperature, 500 °C; and desolvent gas flow rate, 900 L/min. Data were collected by UNIFI 1.8, and mass (Da)-intensity was obtained by deconvolution analysis.
Flow Cytometric Analysis
The nanobodies were tested for binding to Jurkat, U251-MG, AsPC-1, and Panc-1 cells. The cells (1 × 105 per well) were incubated with BsNb PX4, anti-CXCR4 nanobodies, or anti-PD-L1 nanobodies (1 µg/mL). FITC-labelled anti-His secondary mAbs were used. The samples were washed twice with FACS buffer (ice-cold PBS containing 2% FBS), and data were acquired on a CytoFLEX cytometer (Beckman Coulter, California, USA).
PD-L1 and CD171 double-positive U251-MG cells were labelled with CFSE green fluorescent dye (Invitrogen, California, USA), and CXCR4 and CD3 double-positive Jurkat cells were labelled with PKH26 red fluorescent dye (Sigma, Missouri, USA) according to the manufacturer’s protocols. The cells were resuspended and washed three times with FACS buffer. The labelled cells were mixed at an equal ratio (U251-MG cells to Jurkat cells) and then treated with BsNb PX4 for 30 min at 4 °C. The bispecific anti-CD3/CD171 antibody was included as a positive control, and the anti-CXCR4 nanobody was included as a negative control. Flow cytometric analysis was performed to detect CFSE + PKH26 + cells.
Cell migration was evaluated using a Transwell chamber with 5 μm pore filters (Corning Inc., New York, USA). Jurkat cells (2.5 × 105 per well) were added to the upper chambers, and 20 µL of 200 ng/µL hCXCL12 was added to the lower chamber. Jurkat cells were allowed to migrate towards hCXCL12 for 4 h at 37 °C in 5% CO2. To examine antagonistic properties, 0.08 µM BsNb PX4 or the anti-CXCR4 nanobody was added to the upper chamber. To determine the half-maximal inhibitory concentration (IC50), different concentrations of BsNb PX4 were added to the upper chamber. The migrated cells in the lower chamber were collected and quantified by an automated cell counter (Countstar, Shanghai, China).
Analysis of the production of IFN-γ by ELISA
hPBMCs from a healthy volunteer were isolated by Ficoll-Paque® PLUS density gradient centrifugation. T cells were sorted and purified from hPBMCs by using CD3 microbeads (Miltenyi Biotec, Köln, Germany). T cells (6 × 104) were cultured in 96-well plates for 24 h at 37 °C in RPMI 1640 supplemented with 10% FBS in the presence of 50 ng/mL anti-CD3 antibodies and 50 U/mL interleukin-2. After the T cells were activated, 0.3 nM BsNb PX4 and anti-PD-L1 nanobodies were added. Supernatants were collected after 24 h, 48 and 72 h, and the level of IFN-γ was determined by ELISA.
Detection of cell viability and proliferation
To determine the inhibitory effects of the nanobodies, 5 × 103 tumour cells were seeded in 96-well plates and incubated with 0.00001 µM, 0.001 µM and 0.1 µM BsNb PX4 for three days. AsPC-1 cells were plated (5 × 103 per well) in 96-well plates with or without 200 ng/µL hCXCR12. After 4 h at 37 °C, BsNb PX4 and anti-CXCR4 nanobodies were added to a final concentration of 0.1 µM. Cell viability and proliferation were measured using a Cell Counting Kit-8 assay (Dojindo, Tokyo, Japan).
In vitro cytotoxicity assays
Panc-1 or AsPC-1 cells were seeded at 5 × 103 per well overnight in RPMI-1640 medium with 10% FBS before hPBMCs were added at a 10:1 E/T ratio, followed by incubation for 24 h with IL-2 (100 IU/mL). Then, the mixture was cultured for an additional 48 h in the presence of BsNb PX4 (0.5 µM). To examine tumour cell lysis, the release of lactate dehydrogenase (LDH) in the coculture supernatants was measured by a CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, Wisconsin, USA). The percentage of cytotoxicity was calculated as follows: cytotoxicity% = (experimental lysis–spontaneous effector lysis − spontaneous target lysis)/(maximum target lysis − spontaneous target lysis) × 100%.
In vivo activity of bispecific nanobody with hPBMCs
Female NOD/SCID mice aged 6–7 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in a specific pathogen-free room under controlled temperature and humidity conditions. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University. The NOD/SCID mice were subcutaneously injected with 2 × 106 AsPC-1 cells. When the mice bearing AsPC-1 tumours ranging from 50 to100 mm3 were established, 2 × 106 human PBMCs were injected into the tail vein. AsPC-1 cells inoculations and hPBMCs injections were performed under anesthesia with 2% isoflurane inhalation. The mice were randomly divided into four treatment groups: PBS, atezolizumab, BsNb PX4, combination of anti-CXCR4 nanobody and anti-PD-L1 nanobody. The first treatment was administered 1 day after the inoculation of hPBMCs. BsNb PX4 (0.3 mg/kg) or combination of the two nanobodies (anti-CXCR4-VHH at 0.3 mg/kg; anti-PD-L1-VHH at 0.3 mg/kg) was injected intraperitoneally (i.p.) every two days for a total of five times. The positive control drug atezolizumab was administered i.p. at a dose of 3 mg/kg every five days for a total of two times. The tumour volumes were measured every three days and calculated as follows: 0.5×length×width2.
Immunohistochemistry (IHC) and immunofluorescence (IF) staining
All mice were sacrificed by cervical dislocation under anesthesia with 2% isoflurane inhalation before the tumour volume reached 2000 mm3. The tumour grafts were removed, embedded in paraffin and sectioned into 3 μm sections. IHC was performed according to standard protocols. Antigen retrieval was achieved by heating the sections in 0.1 M Tris-HCl buffer (pH 9.0) at 98 °C for 15 min. Endogenous peroxidase was blocked with 3% H2O2 in PBS for 20 min. Subsequently, the sections were blocked with normal goat serum, followed by incubation with primary antibodies (mouse anti-His tag mAbs) at a 1:200 dilution overnight at 4 °C. The sections were stained using a polymer HRP detection system (DAKO, California, USA). The images were captured using an XSP-C204 (COIC, Chongqing, China).
For IF staining, the sections were blocked with 5% BSA for 1 h at room temperature and incubated with CD31 and αSMA antibodies (CST, Massachusetts, USA) for 2 h in the dark. Appropriate DyLight® fluorochrome-conjugated secondary antibodies (Earthox, Massachusetts, USA) were used at 1:200 dilutions for 1 h at 37 °C. After being immunolabelled, the sections were washed and stained with DAPI (Sigma, Mississippi, USA) for 10 min at room temperature. The images were visualized under a NIKON ECLIPSE C1 microscope (NIKON, Tokyo, Japan).
All quantification data are summarized as the mean ± standard deviation (SD) of at least three biological replicates of each experiment. GraphPad Prism 8.0 (GraphPad Software Inc., CA, USA) was used to create the graphs. Standard t tests were used to compare 2 groups, and one-way ANOVA was used to compare more than 2 groups. For all statistical comparisons, P < 0.05 indicated a statistically significant difference.