Antibody and Cell lines
CD20 targeted rituximab (Mabthera) was purchased from Roche. Human lymphoma cell lines (Jurkat and Raji) were used. Both cells were obtained from American Type Culture Collection (ATCC) and maintained in RPMI 1640 with 10% fetal bovine serum (Gibco) containing 1% antibiotics (penicillin G, 100 unitsmL, and streptomycin 10 µgml; Gibco). Cells were incubated at 37 °C in a 5% CO2 atmosphere.
Protein concentrations were determined using a BCA protein assay kit (Thermo Scientific). The membranes were blocked for 1 hour at room temperature and incubated with either anti-CD20 (#sc-58985) or β-actin (#A5441, Sigma-Aldrich) primary antibodies overnight at 4 °C. An enhanced chemical luminescence reagent (Roche) was used, and luminescent signals were measured with a ChemiDoc imaging system (Bio-Rad).
Immunoconjugation of DOTA-rituximab
For the desalting process of rituximab, we exchanged non-metallic D. W (D. W) using Amicon® ultra centrifugal filters (Millipore). The DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid)-NHS ester was purchased from FutureChem (#FC-2134, Seoul, Korea). Briefly, 10 mg rituximab was added to 200 µL of 1 M sodium bicarbonate (pH 8.0) for 5 min. DOTA-NHS (7 nM) was added to the mixture at 4 °C overnight. Excess DOTA-NHS ester was removed using a PD-10 column (GE Healthcare) using 1 mM sodium acetate buffer (pH 5.5). The number of chelators per antibody was calculated by matrix-assisted laser desorption ionisation time-of-ﬂight mass spectrometry (MALDI-TOF MS) to compare the molecular weights of DOTA-rituximab and rituximab.
Radiolabelling and Stability Tests
64CuCl2 was produced at the Korea Institute of Radiological and Medical Sciences (Seoul, Korea) by 50 MeV cyclotron irradiation . The ratio of 64CuCl2 activity per DOTA-rituximab was determined to be 2 MBq per 1 mg. DOTA-rituximab conjugate was incubated with dried 64CuCl2 in 1 mM sodium acetate buffer at 40 °C for 30 min. The radiolabelling yield was evaluated by instant thin-layer chromatography (iTLC) without additional purification. The stability of 64Cu-DOTA-rituximab was analysed through iTLC after incubation in human and mouse serum, and phosphate buffered saline (PBS) at 37 °C for various times (1, 2, 6, 24, 48, and 60 h).
Cell binding assays and immunoreactivity
Cell binding with 64Cu-DOTA-rituximab was performed using Jurkat and Raji cells. Both, Jurkat and Raji cell set-ups (5 × 105 cells /500 µL in tube) were incubated for 3 h in triplicate. Nonspecific binding (competitive inhibition) was performed by adding 11 µM of cold rituximab. After incubation, the cells were rinsed twice with cold PBS containing 1% bovine serum albumin (BSA). Cell bound radioactivity (count per minute) was evaluated using a γ-counter (Wizard 1480, Perkin-Elmer). Specific binding (%) was calculated using total binding and nonspecific binding data. To evaluate immunoreactivity , 3.1 nM of 64Cu-DOTA-rituximab was added to Raji cells diluted from 1 x 107 cells/tube to 0.016 × 107 cells/tube in 500 µL serum-free medium. The incubation time, washing, and calculation methods were the same as above. The immunoreactive fraction was determined by performing a linear regression analysis of the double inverse plot of total/bound activity versus normalised cell concentration. The immunoreactive fraction was then obtained from the inverse of the intercept on the plot. Data analysis was performed using GraphPad Prism software.
Six-week-old female BALB/c-nude mice were obtained from NARA Bio, Inc. All animal experiments were approved by the Institutional Animal Care and Use Committee of KIRAMS (2018-0061). For establishment of lymphoma xenograft mouse models (n=5; for PET/CT imaging, n=3; for biodistribution), Raji cells (5 × 107/ in 200 µL PBS) were transplanted subcutaneously into the right thigh. Small animal PET imaging and biodistribution studies were performed when tumour sizes were > 0.5 cm in diameter.
Small-animal PET/CT imaging
PET/CT images of tumour-bearing mice were acquired using PET/CT (INVEON scanner, Siemens Healthcare). Images were obtained for 20 min under inhalation anaesthesia (isoflurane, 1.5%) at 2, 24, and 48 h after intravenous injection (i.v.) of 7.4 – 7.7 MBq of 64Cu-DOTA-rituximab per mouse. Blocking studies (n=2) were evaluated for CD20 specificity of 64Cu-DOTA-rituximab. Non-labelled rituximab (10 mgmL) was injected intravenously within 2 h before 64Cu-DOTA-rituximab injection. Images were reconstructed with INVEON software and the AMIDE algorithm (A Medical Image Data Examiner).
Biodistribution studies were performed to evaluate the uptake of 64Cu-DOTA-rituximab in tumour-bearing mice or normal mice. All mice were intravenously injected with 1.85 MBq of 64Cu-DOTA-rituximab. Tumour-bearing mice (n=3) were sacrificed 48 h after i.v. injection. For dosimetry, normal mice (n=4/group) were sacrificed at 1, 2, 6, 24, 48, and 72 h after i.v. injection. Various organs containing tumours and blood samples were weighed, and the radioactivity was measured. The γ-counter data were represented by the percentage of injected dose per gram of tissue (%ID/g).
Autoradiography and Immunofluorescence
After the γ-counting of tumours from the biodistribution study, tumour tissues were frozen using optimal cutting temperature (OCT) compound at -80 °C. A cryostat microtome (CM1800, Leica Instruments) was used for frozen sections of tumours tissue (15 μm depth in non-coating slide). The frozen sections were exposed on a film for 7 days in a deep freezer, and the film was scanned with BAS-5000 (Fujifilm). The image intensity of photostimulated luminescence was analysed using Fujifilm Multi Gauge software, version 3.0 (Fujifilm).
For immunostaining, a cryostat microtome was used for frozen sections of the tumours (7 μm depth in coating slide). Briefly, the slides were rinsed using PBS for 10 min and fixed in 4% paraformaldehyde for 10 min. After being washed twice, the slides were incubated in Triton X-100 for 10 min to permeate the tumours tissue. Normal goat serum (1%) was used for non-specific binding and incubated with anti-CD20 (#sc-58985) primary antibody overnight at 4 °C. To visualise specific binding of the antibody, fluorescence-labelled secondary anti-mouse antibody (Bethyl lab) was added to the slides for 1 h. Immunofluorescence images were obtained using IN Cell Analyzer 2200 (GE Healthcare).
Residence time and human extrapolation dosimetry analysis were performed using the OLINDA/EXM software (Organ-Level Internal Dose Assessment/Exponential Modeling computer software, Vanderbilt University, 2003). The residence time was calculated at each time point for 2, 24, 48 h 64Cu-DOTA-rituximab PET/CT acquisition data. The calculated region-of-interest (ROI) was defined based on the CT image. The 225Ac-DOTA-rituximab dosimetry analysis was performed using Monte Carlo simulation. The absorbed dose of 225Ac-DOTA-rituximab also considered all daughter radionuclides (221Fr, 217At, 213Bi, 213Po, 209Tl, and 209Pb) and summed up after applying weighting factors in the two possible pathways, 2% for 209Tl and 98% for 213Po.
S-value and Absorbed dose calculation
The S-value was acquired from tumours and organ tissue density. CT density and PET radioactivity information were used as input data. The S-value and dose map of the tumours and organs were calculated using Geant4 Monte Carlo simulation. The S-value equation is as follows:
where yi is the number of energy, Ei is the energy per radiation, and m is the mass of the target region.
The absorbed dose (DrT) was calculated using the following formula:
where A0 is the initial injected radioactivity and Ãrj is the radiotracer residence time of a source organ ri· S(rT ← rs) is the dose deposited in the target rT per unit of cumulated activity in source rs. The absorbed dose of each organ and tumours was calculated for each organ, including contributions from the self-dose with the cross-dose from other segmented regions .
All statistical analyses were performed using GraphPad Prism software. All data are evaluated as means ± standard deviation (SD) and are representative of at least two separate biological experiments performed in triplicate. Statistical significance between groups was compared using one-way ANOVA and unpaired Student’s t-test. P < 0.05 was considered statistically significant.