Materials, cells and antibodies
), SW626, EL4, HT29, BxPC3, SK-MEL28, and P3X63Ag8.653 cell lines were purchased from ATCC (Manassas, VA). Colo205-luc cells (Bioware® ultra) were obtained from Caliper Life Science (Hopkinton, MA). The murine control mAb 121SLE (IgM) was purchased from GeneTex (Irvine, CA). Sialyl Lewis A tetrasaccharide (Cat # S2279) was purchased from Sigma-Aldrich (St. Louis, MO). sLea
-HSA conjugate (Cat # 07-011), monovalent biotinylated-sLea
-sp-biotin; Cat # 02-044), polyvalent biotinylated sLea
-PAA (Cat # 01-044), biotin-labeled Lea
-PAA (Cat # 01-035) and sLex
-PAA-biotin (Cat # 01-045) were purchased from GlycoTech (Gaithersburg, MD). In the polyvalent presentation, the tetrasaccharide is incorporated into a polyacrylamide matrix (PAA) thereby creating a 30kd multivalent polymer with approximately every 5th amide group of the polymer chain N- substituted with biotin in a 4:1 ratio and approximately 20% carbohydrate content. Other HSA or BSA glycoconjugates used in this study were prepared in house using sLea
pentenyl glycoside as described (11
). GD3, fucosyl-GM1, GM2 and GM3 were purchased from Matreya (Pleasant Gap, PA) and GD2 was purchased from Advanced ImmunoChemical (Long Beach, CA).
Generation of anti-sLea mAb producing hybridomas
Blood samples were obtained from 3 patients in an ongoing trial with sLea
-KLH conjugate vaccine in patients with breast cancer initiated at MSKCC under an MSKCC and FDA approved IRB protocol and IND. Blood specimens were selected from 2 patients after 3 or 4 vaccinations, which showed antibody titers against sLea
of 1/160 and 1/320, respectively. These sera (and murine mAb 19.9) react well with sLea
positive cell lines in FACS assays and mediate, potent CDC (11
). PBMCs were isolated from approximately 80-90 ml of blood by gradient centrifugation on Histopaque-1077 (Sigma-Aldrich).
PBMCs were cultured in RPMI-1640 medium (Mediatech, Manassas, VA) supplemented with L-Glutamine, non-essential amino acids, sodium pyruvate, vitamin, penicillin/streptomycin, 10%FBS (Omega Scientific, Tarzana, CA), 10 ng/ml IL-21 (Biosource, Camarillo, CA), and 1μg/ml anti-CD40 mAb (G28-5 hybridoma supernatant, ATCC). Cells were fused by electrofusion to P3X63Ag8.653 myeloma cells.
For the sLea ELISA, plates were coated either with 1 μg/ml of sLea-HSA conjugate, monovalent biotinylated-sLea, or with polyvalent biotinylated sLea-PAA captured on Neutravidin (Pierce, Rockford, IL) coated plates. Uncoated wells (PBS) and human serum albumin (HSA) coated wells were used as controls. Bound antibodies were initially detected with HRP-labeled goat anti-human IgA+G+M (Jackson ImmunoReseach, West Grove, PA) and positive wells were subsequently probed with IgG-Fc or IgM specific secondary antibodies to determine isotypes.
Carbohydrate Specificity Analysis
Cross-reactivity against the closely related antigens, Lea and sLex, was evaluated by Surface Plasmon Resonance (SPR) and confirmed by ELISA using biotin-labeled Lea-PAA and biotin-sLex-PAA. Binding to gangliosides GD2, GD3, fucosyl-GM1, GM2, and GM3 was tested by ELISA. A competition ELISA was used to evaluate the specificity of the mAbs against several other related carbohydrate moieties. In brief, 2 μg/ml sLea-HSA conjugate was coated onto plates followed by blocking with 3%BSA in PBS. Next, 30 μl of different carbohydrate moieties (40 μg/ml in PBS prepared from 1 mg/ml stock solutions) either unconjugated or conjugated to HSA or BSA was mixed each separately with 30 μl of test antibody and incubated at room temperature in a sample plate. After 30 minutes 50 μl of the mixture was transferred to the coated assay plate and incubated for 1 hr, followed by incubation with HRP-labeled goat anti-human IgA+G+M, washing and colorimetric detection of bound antibody using a Versamax spectrofluorometer (all steps are performed at room temperature). The tested carbohydrate moieties included globo H, Lewis Y, Lewis X, sialyl-Thomson-nouveaux (sTn), clustered sTn, Thomson Friedenreich (TF), Tighe Leb/LeY mucin, porcine submaxillary mucin (PSM), as well as sLea tetrasaccharide and sLea-HSA conjugate. To determine the fine specificity of the antibodies, glycan array analysis was performed by the Consortium for Functional Glycomics Core H group. 5B1 and 7E3 antibodies were tested at 10 μg/ml using version 4.1 of the printed array consisting of 465 glycans in replicates of 6.
Immunoglobulin cDNA cloning and recombinant antibody expression
Variable region of human mAb heavy and light chain cDNA was recovered by RT-PCR from the individual hybridoma cell line and subcloned into IgG1 or IgM heavy chain, or IgK or IgL light chain expression vector as described before (13
). Ig heavy chain or light chain expression vector were double digested with Not I and Sal I, and then both fragments were ligated to form a dual gene expression vector. CHO cells in 6 well-plates were transfected with the dual gene expression vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 24 hrs, transfected cells were transferred to 10 cm dish with selection medium [D. MEM supplemented with 10% dialyzed FBS (Invitrogen), 50 μ M L-methionine sulphoximine (MSX), GS supplement (Sigma-Aldrich) and penicillin/streptomycin (Omega Scientific, Tarzana, CA)]. Two weeks later MSX resistant transfectants were isolated and expanded. High anti- sLea
antibody producing clones were selected by measuring the antibody levels in supernatants in a sLea
specific ELISA assay and expanded for large scale mAb production.
Human mAb purification
Antibodies were purified using the Äkta Explorer (GE Healthcare, Piscataway, NJ) system running Unicorn 5.0 software. In brief, stable clones of 5B1 or 7E3, respectively, were grown in serum-free culture medium in a Wave™ bioreactor and the harvested supernatant was clarified by centrifugation and filtration and stored refrigerated until used. Human IgG antibodies were purified on appropriate sized Protein A columns using 10 mM PBS, 150 mM NaCl running buffer. Human IgM antibodies were purified on a hydroxyapatite column, and IgM was eluted with a gradient of 500 mM phosphate. The antibody concentrations were determined by OD280 using an E1% of 1.4 and 1.18 for IgG and IgM, respectively, to calculate the concentration. The purity of each preparation was evaluated by SDS-PAGE analysis (1-5 μg/lane) under reducing conditions and the purity was estimated > 90% based on the sum of heavy and light chains.
positive or negative tumor cell lines (0.5×106
cells per condition) were washed in PBS/2% FBS (PBSF). Test or control human mAb was then added (1-2μg/ml in complete medium) and incubated on ice for 30 minutes (14
). After washing in PBSF, the cells were incubated with Alexa-488 anti-human IgG (Fcγ) or anti-human IgM (μ) (Invitrogen) for 30 minutes on ice. Cells were washed twice in PBSF and analyzed by flow cytometry using the Guava Personal Cell Analysis-96 (PCA-96) System (Millipore, Billerica, MA). Colo205-luc cells were incubated with 2 μg/ml primary antibody followed by staining with secondary antibodies from SouthernBiotech (Birmingham, AL), and analyzed on a Becton Dickinson FACS Advantage IV instrument using FlowJo 7.2.4 software.
Affinity constants were determined using the principal of Surface Plasmon Resonance (SPR) with a BiaCore 3000 (GE Healthcare, Piscataway, NJ). Biotin-labeled univalent sLea (Cat # 02-044) or polyvalent sLea-PAA-biotin (Cat # 01-044) were coupled to separate flow cells of an SPA biosensor chip according to the manufacturer’s instructions. A flow cell blocked with HSA and culture medium containing free biotin was used as a reference cell. The binding kinetic parameters were determined from several known concentrations of antibody diluted in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20) using the sLea-PAA-biotin coated flow cell. The curve-fitting software provided by the BiaCore instrument was used to generate estimates of the association and dissociation rates from which affinities are calculated.
Complement dependent cytotoxicity (CDC) assay
antigen positive and negative cell lines were used with a 90 minute cytotoxicity assay (Guava PCA-96 CellToxicity™ kit, Millipore, Billerica, MA, Cat # 4500-0200) using human complement (Quidel, San Diego, CA, Cat # A113) and purified human mAb at various dilutions (0.1 – 25 μg/ml) or with positive control mAbs as previously described (15
). In brief, 2.5×106
target cells were painted with carboxyfluorescein diacetate succinymyl ester (CSFE) to yield green/yellow fluorescent target cells. The painted cells (1×105
/50 μl sample) were incubated for 40 minutes with 100 μl of antibodies on ice. Next, 50 μl of human complement diluted 1:2 in complete medium (RPMI-1640, 10% FCS) or medium alone was added to triplicate samples and incubated for 90 minutes at 37°C. Thus, the final complement dilution in the assay was 1:8. Cells that were killed during this incubation time were labeled by adding the membrane impermeable dye 7-aminoactinomycin D (7-AAD) and samples were analyzed by dual color immunofluorescence utilizing the Guava CellToxicity™ software module. Control samples that receive NP40 were used to determine maximal killing and samples receiving complement alone served as baseline. The percentage of killed cells was determined by appropriate gating and calculated according to the following formula: % killed = (% sample – % complement alone) / (%NP40 - % complement alone)x100.
Antibody dependent, cell mediated cytotoxicity (ADCC) assay
PBMC effector cells were isolated from blood samples obtained under an MSKCC IRB approved protocol by Ficoll-Hypaque density centrifugation. The target cells were incubated at 5×106 cells/ml in complete growth media with 15 μl of 0.1% calcein-AM solution (Sigma-Aldrich) for 30 minutes at 37°C, in the presence of 5% CO2. The cells were washed twice with 15 ml of PBS-0.02% EDTA and resuspended in 1ml complete growth medium. Fifty μl (10,000 cells) of labeled target cells were plated into a 96 well plate in the presence or absence of antibodies at the concentrations described in , and incubated with 50 μl of freshly isolated peripheral blood mononuclear cells (effector cells, at 100:1 E/T ratio) accordingly. After 2 hours incubation, the plate was centrifuged at 300xg for 10 minutes and 75 μl of supernatant was transferred into a new flat bottomed 96 well plate. The fluorescence in the supernatant was measured at 485nm excitation and 535nm emission in Fluoroskan Ascent (Thermo Scientific). Spontaneous release was determined from target cells in RPMI-1640 medium with 30% FBS without effector cells and Maximum release was determined from target cells in RPMI-1640 medium with 30% FBS and 6% Triton X-100 without effector cells. Percent cytotoxicity was calculated as [counts in sample-spontaneous release]/[maximum counts-spontaneous release]x100.
ADCC activity of r5B1 antibodies
mAb internalization assay
Internalization of 5B1 antibody was evaluated by measuring the cytotoxic activity of r5B1 and Hum-ZAP secondary conjugate (Advanced Targeting Systems, San Diego, CA) complex against sLea expressing BxPC3 cells. BxPC3 cells were plated into a 96 well plate (2,000 cells/90μl/well) and incubated overnight in duplicates. Various concentrations of 5B1 antibody were incubated with Hum-ZAP secondary conjugates at RT according to the manufacturer’s instruction. Next, 10 μl/well of r5B1 and Hum-ZAP complex was added to the cells and incubated for 3 days. Twenty five μl of Thiazolyl Blue Tetrazolium Bromide (Sigma-Aldrich) solution (5mg/ml in PBS) was added to each well and incubated at 37°C. After a 2hr incubation 100μl/well of solubilization solution (20% SDS/50% N,N-Dimethylformamide) was added to each well and incubated for another 16 hrs at 37°C. The OD was measured at 570/690nm and values obtained with medium alone were used for plate background subtraction. Eight parallel cultures without antibody were used to normalize the sample values (Sample/Mean Untreated*100).
Xenograft transplantation model
Female CB17 SCID mice (5-8 weeks old) were purchased from Taconic (Germantown, NY). Colo205-luc cells (0.5 × 106) in 0.1 ml complete growth media were injected via the tail vein on Day 0 using a BD insulin syringe with 28G needle (BD, Franklin Lakes, NJ). One hundred μg of mAb 5B1 was injected intraperitoneally on days 1, 7, 14 and 21 (experiment 1), or on days 1, 4, 7, 10, 14 and 21 (experiment 2). Mice were monitored for tumor development. All procedures were performed under a protocol approved by the Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee. Kaplan-Meier survival curves were generated using GraphPad Prism 5.1 (GraphPad Software, San Diego, CA) and analyzed using the Mantel-Haenszel log-rank test.