Engineering anti-Lewis-Y hu3S193 antibodies with improved therapeutic ratio for radioimmunotherapy of epithelial cancers

The construction and production of hu3S193 and huA33 has been described before [31, 32]. The hu3S193 heavy chain (HC) was ligated into the pEE6.4 mammalian expression vector (Lonza Biologics, Slough, UK) via a HindIII and EcoRI double digest (pEE6.4/hu3S193 HC). pEE6.4/hu3S193 HC was used as a template for site-directed mutagenesis (GeneTailor™ Site-Directed Mutagenesis System (Invitrogen) or QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene) to introduce the following substitutions in the CH2 and CH3 domains: I253A, H310A, H435A and I253A/H310A [25]. The hu3S193 kappa light chain was ligated into the pEE14.4 mammalian expression vector containing the glutamine synthetase gene (Lonza Biologics) via a HindIII and EcoRI double digest. For the simultaneous expression of each mutated hu3S193 antibody, light and mutated heavy chain genes were cloned into a double-gene vector (pDGV) using a NotI and PvuI double digest. Freestyle 293-F cells (1 × 10⁶ cells/mL) were transfected with the pDGV constructs according to the manufacturer’s instructions (Invitrogen). Supernatants were harvested at 96 h post transfection. Hu3S193 antibodies were purified using HiTrap KappaSelect columns (GE Healthcare). Purified proteins were analysed by SDS-PAGE under non-reducing conditions and evaluated by size exclusion chromatography on a Superdex 200 HR 10/30 column (GE Healthcare) using 0.01 mol/L sodium phosphate and 0.15 mol/L NaCl (pH 7.2) as elution buffer.

The Lewis-Y binding activity of hu3S193 antibodies was determined by BIAcore analysis using a synthetic Lewis-Y tetrasaccharide coupled to BSA (Alberta Research Council, Edmonton, Alberta, Canada) on a CM5 chip using a BIAcore 2000 as described (BIAcore AB, Uppsala, Sweden) [30]. Hu3S193 antibody samples were diluted in HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM di-Na-EDTA and 0.005 % Tween 20). Aliquots (60 μL) were injected over the sensor chip surface at a flow rate of 30 μL/min. After the injection phase, dissociation was monitored by flowing HBS over the chip surface for 300 s. Bound antibody was eluted, and the chip surface was regenerated between samples by injection of 100 mM HCl. For kinetic analysis, varying concentrations of hu3S193 wild type and mutants were injected (10, 19, 38, 75 and 150 nmol/L). Global analysis using a 1:1 Langmuir model fit was performed using BIA-evaluation version 4.1.1 software.

Fluorescence-activated cell sorter (FACS) analysis was done on Lewis-Y-positive A431 skin cancer cells. Aliquots of 2 × 10⁵ cells were incubated with hu3S193 antibodies (200 nmol/L) in DMEM/F12 medium containing 10 % foetal bovine serum on ice. After washing the cells with PBS, cells were incubated with a goat phycoerythrin-conjugated anti-human IgG (Sigma) and incubated on ice for 30 min. Cells were washed with phosphate-buffered saline and resuspended in a final volume of 300 μL. In control samples, primary antibody was omitted. Flow cytometric analysis was done using a Guava EasyCyte Plus flow cytometer. Cancer cell populations were gated based on forward and side scatter variables. Data analysis was done using WinMDI (Joseph Trotter).

Hu3S193 antibodies were radiolabelled with four isotopes: ¹²⁵I, ¹³¹I, ¹¹¹In and ¹⁷⁷Lu. Iodine-125 and lutetium-177 were obtained from PerkinElmer (PerkinElmer Life and Analytical Sciences, Waltham, MA), iodine-131 was obtained from ANSTO (ANSTO, Menai, Australia) and indium-111 was obtained from MDS Nordion (Canada).

Radioiodination was performed using pH-neutralised isotopes, catalysed by iodogen-coated glass beads as previously published [12, 33]. After a 10-min incubation period, the reaction mixture was purified through a Sephadex G50 desalting column (Sigma-Aldrich, Sydney, Australia) equilibrated with 0.9 % NaCl containing 0.05 % human serum albumin.

Radiolabelling of the hu3S193 antibody constructs with indium-111 and lutetium-177 was done by using the bifunctional metal ion chelate C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A″ DTPA) [34, 35]. A chelate to antibody ratio of 3:1 was employed, using 0.1 M sodium bicarbonate buffer (pH 8.6) containing 0.9 % NaCl. Incubation was allowed for 16 h at room temperature. Under these conditions, one to two chelates are expected per antibody molecule. The radiolabelled mixture was purified through a Sephadex G50 desalting column equilibrated with 0.9 % NaCl containing 0.05 % human serum albumin.

Radiolabelling was performed on the day of injection into mice. Prior to injection, the percentage of unbound radionuclide content was determined by ITLC as previously described [36]. Determination of the immunoreactivity of radiolabelled hu3S193 antibody constructs was performed by a single-point binding assay, where 10 × 10⁶ Le^y-positive A431 cells were incubated with 20 ng of radiolabelled antibody constructs for 45 min at room temperature with continuous mixing throughout to keep the cells in suspension. Cells were washed three times, and pellets were measured in a gamma counter (Cobra II, Model 5002, Packard Instruments, Canberra, Australia). Three samples of radiolabelled antibody at the same concentration as that initially added to the cells were measured at the same time of the cell pellets, and immunoreactivity was calculated: (cpm cell pellet/mean cpm radioactive antibody standards) × 100. Serum stability was analysed by determination of immunoreactivity on the day of injection, at 48 h, and 7 days after 20 ng of radiolabelled antibody was incubated in human serum at 37 °C.

Female athymic mice (BALB/c nu/nu; 4–6 weeks; Animal Resources Centre) were injected in the tail vein with 0.185 MBq ¹²⁵I-mutant (2.5–5 μg, 5 μCi) and 0.185 MBq ¹³¹I-hu3S193 (2.5–5 μg, 5 μCi) wild type antibody (n = 5). A separate group of athymic mice were injected with 0.185 MBq ¹²⁵I- hu3S193 mutant (2.5–5 μg, 5 μCi) and 0.185 MBq ¹¹¹In-hu3S193 (2.5–5 μg, 5 μCi) mutant (n = 5). All animal studies were approved by the Austin Hospital Animal Ethics Committee and were conducted in compliance with NHMRC Australian code of practice for the care and use of animals for scientific purposes. Blood samples (10–20 μL) were collected from groups of five at 5 min, 1, 2, 4, 8, 24, 48, 72, 120, 168, 240 and 336 h after injection of radioactive antibodies. Samples were counted in a gamma counter (Cobra II). Standards prepared from injected material were counted each time with blood samples enabling calculations to be corrected for physical decay of isotope.

Biodistribution studies were done with athymic non-tumour-bearing mice (BALB/c nu/nu, female, 4–6 weeks) or mice bearing A431 tumours (¹¹¹In, 0.657 ± 0.216 g; ¹³¹I, 0.515 ± 0.195 g; ¹⁷⁷Lu, 0.466 ± 0.086 g). To study the tumour uptake and biodistribution in normal tissues of radioiodinated hu3S193 variants, A431 tumour-bearing BALB/c nu/nu mice were co-injected intravenously with 0.185–0.74 MBq (5–20 μCi) ¹³¹I-hu3S193 variants (2–6 μg protein) and 0.185–0.74 MBq ¹²⁵I-hu3S193 wild type (2–6 μg protein). In a second study, 0.185–0.74 MBq ¹¹¹In-CHX-A″ DTPA-labelled mutant (2–6 μg protein) and 0.185-0.74 MBq ¹²⁵I-labelled hu3S193 wild type (2–6 μg protein) were co-injected. ¹¹¹In-CHX-A″ DTPA-labelled wild type and ¹²⁵I-hu3S193 wild type were also injected as a control group. ¹²⁵I-hu3S193 wild type was used as an internal control in all injected animals to enable direct comparison between the different mutants. Typically, groups of four to five mice were sacrificed at 4, 24, 48, 72, 120, 168, and 240 or 288 h after injection of radiolabelled antibodies. For evaluation of ¹⁷⁷Lu-labelled antibodies, mice were sacrificed at 48 h after injection. At the designated time points, groups of mice (n = 4–5) were humanely sacrificed by over-inhalation of isoflurane. Mice were bled by cardiac puncture, and tumours and organs (skin, liver, spleen, small intestine, stomach, kidney, brain, bone (femur), lungs and heart) were immediately removed and blotted dry. All samples were weighed and counted in a dual gamma scintillation counter (Cobra II, Packard Instruments). Triplicate standards prepared from the injected material were counted at each time point with tissue and tumour samples enabling calculations to be corrected for the physical decay of the isotopes. Results of the radiolabelled antibody distribution over time were calculated as the mean percentage injected dose per gramme (%ID/g ± SD) for each mutant and parental hu3S193 per time point.

Antibody serum concentrations were expressed as percentage injected dose per millilitre (%ID/mL), and the blood concentrations (μg/mL) were calculated. A two-compartment IV bolus model with macro-parameters, no lag time and first order elimination (WinNonlin Model 8) was fitted to serum data obtained from blood clearance studies for each animal using unweighted non-linear, least squares with WinNonLin version 5.2 (Pharsight Corp., Mountain View, CA). Estimates were determined for the pharmacokinetic parameters: alpha half-life (t _1/2α), beta half-life (t _1/2β), area under the curve extrapolated to infinity (AUC_0-∞) and mean residence time (MRT). Alpha half-lives of mutant hu3S193 were constrained to be smaller or equal to the alpha half-life of hu3S193 wild type. Significant differences in these values were examined by comparing the coefficient of variation (CV %) for the estimated parameters.

To estimate radioimmunotherapeutic applications for ⁹⁰Y- or ¹⁷⁷Lu-labelled mutants, biodistribution data obtained with ¹¹¹In-CHX-A″ DTPA-labelled antibodies were used to generate time-activity curves for the calculations of predictive radiation doses for the bone marrow, liver, kidneys and tumour. Identical biodistribution and biologic clearance of ¹¹¹In-, ⁹⁰Y- and ¹⁷⁷Lu-labelled antibodies were assumed. Predictive dosimetric analysis was also done for ¹³¹I-labelled antibodies based on biodistribution data obtained with ¹²⁵I-labelled antibodies. As radiation absorbed doses are proportional to %ID/g, time-activity curves for the blood, liver, kidneys and tumour were integrated over time to calculate area under the curve (AUC). Time-activity curves generated from biodistribution data were corrected for radiodecay. Therefore, the pharmacokinetic values calculated from such data refer to pharmacological values of the antibodies in the absence of radioisotope. Appropriate physical half-life corrections were applied to convert %ID_{pharmacological}/g to %ID_radioisotope/g, and the time-activity curves were fit to either a two or three exponential function from which the AUC is determined for ⁹⁰Y, ¹⁷⁷Lu and ¹³¹I. AUC integration from zero to infinity was done by the sum of a trapezoidal integration of the measurement range (AUC_0–288 h) plus an extrapolated model fit for the extrapolated range (AUC_288 h–∞). To calculate accumulated activity, photon dose and edge effects were ignored. Dose to red marrow was determined from blood concentrations using a baseline value of 0.1 [21].

All the SPECT and MR scans were performed on a small animal nano-SPECT/CT imaging system and a small animal nano-PET/MR imaging system (Mediso nano-Scan^PM, Mediso Medical Imaging Systems, Budapest, Hungary) individually. Groups of two mice were injected with 3.7 MBq (270 μg) ¹⁷⁷Lu-CHX-A″ DTPA-labelled antibody (huA33 wild type, hu3S193 wild type or hu3S193 I253A/H310) and serially imaged at 2 days post injection. Imaging procedures involved anaesthesia of mice by isoflurane. Each mouse was scanned in supine position with its head secured via ear and tooth bars. Respiration was monitored by a pressure-sensitive pad adhered to the abdomen. The imaging study started with a whole body T1-weighted MR scan, followed by a 60-min SPECT scan which was reconstructed into a volumetric image with the voxel size of 0.3 mm × 0.3 mm × 0.3 mm using the Mediso Tera-TOMO® Monte Carlo-based algorithm. Subsequently, reconstructed SPECT and MR images were transferred to a research PACS system where the images could be retrieved for further processing and analysis.

To confirm data obtained from imaging, groups of five mice bearing A431 tumours were injected with 6.29 MBq (100 μg) ¹⁷⁷Lu-CHX-A″ DTPA-labelled antibody (huA33 wild type, hu3S193 wild type or hu3S193 I253A/H310) and sacrificed at 48 h post injection. Organs and blood were collected as described before, and the radiolabelled antibody distribution over time was calculated as the mean %ID/g ± SD for each antibody per time point.

To compare tumour uptake at different time points for the mutants versus hu3S193 wild type, two-way ANOVA with the Bonferroni post test was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA www.​graphpad.​com. Tumour-to-tissue ratios or tissue uptake at specific time points were analysed using one-way ANOVA with Tukey’s multiple comparison post test. When less than three groups were compared, a non-parametric t test (one-tailed) was used.