The humanized MUC1 antibody (HzMUC1) was generated as described previously . Briefly, to obtain the humanized IgG1 antibody with intact IgG format, DNAs encoding VH and VL were synthesized (Tsingke, China) and inserted into the modified pcDNA 3.4 expression vector (Thermo Fisher Scientific, Waltham, MA, USA) carrying the human IgG1 constant region (CH1-hinge-CH2-CH3) or the human kappa chain constant region (CL). These vectors were transiently co-transfected into HEK-293 F cells. After 6 days, HzMUC1 antibody was purified from culture supernatants using Protein-A chromatography (GE Healthcare Life Sciences, Buckinghamshire, UK).
To detect MUC1 protein in cells by western blotting and immunoprecipitation, commercially available rabbit monoclonal antibody against the cytoplasmic tail of MUC1 (CT) (anti-MUC1-CT Ab, Catalog No. ab109185 at 1:5000) was purchased from Abcam (Cambridge, UK). Anti-MUC1 mouse monoclonal antibody to MUC1-N (clone GP1.4; Catalog No. AM32842PU-S at 1:2000) was obtained from OriGene (Rockville, USA). The anti-β-actin antibody (Catalog No. 3700 S at 1:5000), anti-Ki-67 antibody (Catalog No. 9027 S at 1:400), anti-cleaved caspase-3 antibody (Catalog No. 9661 S at 1:1000), anti-caspase-3 antibody (Catalog No. 9662 S at 1:1000) were purchased from Cell Signaling (Danvers, MA, USA).
Human pancreatic cancer cell lines Capan-2, CFPAC-1, SW1990, Mia-PaCa-2, PATU-8988, PANC-1, and Normal pancreatic duct cell line hTERT-HPNE were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Capan-2 cells were cultured in McCoy’s 5 A medium (Thermo Fisher Scientific). CFPAC-1 cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Thermo Fisher Scientific). SW1990, Mia-PaCa-2, PATU-8988, PANC-1, and hTERT-HPNE cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific). All media were supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), penicillin (100 U/mL), and streptomycin (100 µg/mL). Cells were incubated at 37 °C in an atmosphere of 5% CO2. HEK-293 F cells were cultured in suspension with serum-free medium (Catalog No. 12338018; Thermo Fisher Scientific), and incubated at 37 °C in an atmosphere of 8% CO2.
Immunoblotting and immunoprecipitation
For immunoblotting, cell lysates were boiled and separated by SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, MA, USA), and blocked with 5% non fat skim milk in TBS-T for 1 h at room temperature. The membranes were probed with primary and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and developed using chemiluminescence (ECL) reagent. The immunoreactive proteins were detected using ChemiDoc MP imaging system (Bio-Rad) and analyzed with Image Lab 5.0 software (Bio-Rad).
For immunoprecipitation, cells were lysed in 1% NP-40 lysis buffer containing protease inhibitor cocktail mixtures (Selleck Biosciences, San Jose, CA). Cell lysates were incubated with HzMUC1 antibody overnight at 4ºC and followed by incubation with Protein A bead for an additional 1 h. The immunocomplexes were washed with lysis buffer and denatured with Laemmle buffer, followed by immunoblotting with anti-MUC1-CT antibody as described previously.
Pancreatic cancer cells were seeded on poly-L-lysine-coated glass coverslips in 24-well culture plates. After 72 h, cells were blocked with 3% BSA, and incubated with HzMUC1 antibody for 1 h on ice, washed with PBS containing 1% BSA, and incubated with Alexa Flour 488-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch) for 30 min. All samples were mounted and observed under the Nikon A1 confocal microscope system (Nikon, Tokyo, Japan).
Generation of antibody-drug conjugate
The HzMUC1-MMAE and human IgG-MMAE ADC were generated as described previously . Briefly, HzMUC1 or human IgG (Bethyl Laboratories, Montgomery, TX, USA) were partially reduced with tris (2-carboxyethyl) phosphine hydrochloride (TCEP, Sigma-Aldrich St. Louis, USA) at 37 °C for 2 h, and the buffer was exchanged by passing the mixture through Sephadex G25 resin and eluted with PBS. The drug-linker agent (mc-vc-PAB-MMAE, MCE, Monmouth, NJ, USA) dissolved in DMSO was then added to the reduced antibody at 4 °C for 1 h. The reaction mixture was concentrated by centrifugal ultra-filtration, passed through Sephadex G25, and eluted. The eluate was then sterile filtered through a 0.2-µm filter and stored 4 °C.
Colony formation assay
Cells were seeded in 48-well tissue culture plates (3 × 103 cells/well), and treated with the indicated concentrations of HzMUC1 antibody, human IgG-MMAE, and HzMUC1-MMAE. After 5 days, colonies were fixed with 4% paraformaldehyde solution, and stained with 0.5% crystal violet solution at RT for 20 min. Stained crystal violet dye was solubilized with 10% acetic acid. Absorbance was measured at 540 nm using a microplate reader (Molecular Devices, San Jose, CA). The inhibitory concentration (IC50) for HzMUC1-MMAE was determined using SPSS statistics software.
Cell cycle analysis
Pancreatic cancer cells were treated with HzMUC1-MMAE for 24 h, harvested by trypsinization, washed with cold PBS, and fixed with cold 70% ethanol at 4 °C for 2 h. The fixed cells were washed with PBS and incubated with Propidium iodide (Dojindo, Kumamoto, Japan) at 37 °C in the dark for 30 min and detected by Flow cytometry (NovoCyte™, ACEA Biosciences, Hangzhou, China) and analyzed using the NovoExpress software.
Cells were seeded in 6-well culture plates, and treated with the indicated concentrations of human IgG-MMAE and HzMUC1-MMAE. After 72 h, cells were harvested with trypsin-EDTA and washed with FACS buffer. The apoptotic cells were stained with annexin V and propidium iodide (Dojindo) for 15 min at RT in the dark, and analyzed via Flow cytometry (NovoCyte™, ACEA Biosciences) using the NovoExpress software.
Xenograft tumor studies
The experimental protocol was approved by the by the Institutional Animal Care and Use Committee of Wenzhou Medical University (Permit Number: 2020-015). Capan-2 cells (3.5 × 106) or CFPAC-1 cells (3 × 106) were resuspended in 50% Matrigel (Corning, Bedford, MA, USA) and injected subcutaneously into the dorsal right flank of six-week old female BALB/c nu/nu mice (purchased from GemPharmatech, Jiangsu, China) .When the tumors grew up to about the size of 120 ~ 150 mm3, mice were randomly divided into two groups, PBS and HzMUC1-MMAE (5 mg/kg). PBS or ADCs were injected intravenously into the mice every six days for three times. Tumor diameters were measured with calipers, and tumor volumes were calculated using the formula, V = width2 × length/2. Mice were sacrificed on the 18th (Capan-2 tumor) or 21st day (CFPAC-1 tumor) after ADCs injection. On day 18 ~ 21 after ADCs injection, all mice were euthanized, and the tumors were surgically excised, weighed, fixed in 10% formalin, and embedded in paraffin.
Paraffin blocks of tumor were sliced into 4 μm-thick sections and dried onto slides. The sections were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide for 30 min. For antigen retrieval, slides were immersed in citrate solution (pH 6.0) heated at 121 °C for 20 min, and then blocked with 3% normal horse serum for 30 min. The slides were incubated with anti-Ki-67 antibody (1:400) or anti-cleaved caspase-3 antibody (1:100) overnight at 4 °C followed by incubated with HRP-conjugated anti-rabbit IgG antibody (ZSGB-BIO, Beijing, China) for 1 h. The immunoreactivities were detected with 3,3′-diaminobenzidine (DAB, ZSGB-BIO) and examined under an Eclipse Ci microscope (Nikon, Japan).
All data are presented as mean ± standard deviation. Statistical significance of differences between two samples was performed using unpaired two-tailed Student’s t-test. One-way ANOVA with Bonferroni’s multiple comparison test correction was used to analyze data among multiple groups. Statistics were performed using Prism (Graph Pad Software Inc). Statistically significance was considered when p-value was less than 0.05. All experiments were repeated at least three times.