Preclinical study of ²¹²Pb alpha-radioimmunotherapy targeting CD20 in non-Hodgkin lymphoma

Abstract

Background Despite therapeutic advances, Non-Hodgkin lymphoma (NHL) relapses can occur. The development of radioimmunotherapy (RIT) with α-emitters is an attractive alternative. In this study, we investigated the potential of α-RIT in conjunction with ²¹²Pb-rituximab for the treatment of NHL.

Methods EL4-hCD20-Luc cells (mouse lymphoma cell line) were used for in vitro and in vivo studies. Biodistribution and efficacy studies were performed on C57BL/6 mice injected intravenously with 25 × 103 cells.

Results ²¹²Pb-rituximab (0.925–7.4 kBq/mL) inhibit proliferation of EL4-hCD20-Luc cells in vitro. Biodistribution of ^203/212Pb-rituximab in mice showed a significant tumour uptake and suggested that the liver, spleen, and kidneys were the organs at risk. For efficacy studies, mice were treated at either 11 days (early stage) or 20–30 days after injection of tumour cells (late stage). Treatment with 277.5 kBq ²¹²Pb-rituximab significantly prolonged survival. Even at an advanced tumour stage, significant tumour regression occurred, with an increase in the median survival time to 28 days, compared with 9 days in the controls.

Conclusions These results show the efficacy of ²¹²Pb-rituximab in a murine syngeneic lymphoma model, in terms of significant tumour regression and increased survival, thereby highlighting the potency of α-RIT for the treatment of NHL.

Methods:

Cell lines, antibodies, and mice The murine EL4-hCD20-luc lymphoma cell line [14] was maintained in supplemented RPMI 1640 medium. CD20 expression was verified by flow cytometry (Accuri® C6; BD Biosciences, Franklin Lakes, NJ, USA) and bioluminescence imaging (BLI) (IVIS® Lumina LT III; PerkinElmer, Waltham, MA, USA). Rituximab, a mouse/human chimeric mAb (IgG1/kappa) against human CD20, was purchased from Roche (Bâle, Switzerland). For the IgG isotypic control, palivizumab (AbbVie, North Chicago, IL, USA), a humanised mouse mAb (IgG1/kappa) against an epitope of the F protein of respiratory syncytial virus was used. C57BL/6 mice (female, 7–10 weeks) were purchased from Janvier Labs (Le Genest-Saint-Isle, Mayenne, France).

Tumour model C57BL/6 mice (8 weeks old) were injected intravenously in the tail with 25 × 103 EL4-hCD20-luc cells (in 100 µL phosphate-buffered saline [PBS]). Mice were monitored daily for signs of pain or discomfort and three times a week to evaluate and grade facial expressions of pain [15], behaviour, hair coat scruffiness, weight loss, tumour, and mobility. BLI was performed twice a week after 15–20 days (appearance of detectable tumour). Biodistribution experiments were performed 25 days after engraftment. RIT was performed either 11 days post-engraftment or once the tumour was detectable by BLI (20–30 days post-engraftment).

Independent groups of animals were used for the different studies. All in vivo experiments were performed in accordance with animal ethics legislation, and all efforts were made to minimise suffering e.g., by adding nutritional hydrogel. Animals sample size were chosen to minimise the number of animals used in accordance with ethical rules and to respect the principal of the 3Rs. The protocol was approved by the national agency of research (Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation - APAFIS#15900-201807061621591 v2).

In vivo BLI Before in vivo BLI, mice were anaesthetised by isoflurane inhalation and then shaven as well as depilated on the ventral and dorsal faces. The mice were injected with 2 mg VivoGloTM luciferin (Promega, Madison, WI, USA). Imaging was performed 11 min post-injection using the IVIS® Lumina LT III in vivo imaging system (PerkinElmer) with a binning factor of 16 and an automatic exposure time. Living Image® 4.5 software (PerkinElmer) was used to analyse the signal intensities in longitudinal studies. The different acquisitions were normalised on the same scale.

mAb conjugation and radiolabeling The two IgGs were conjugated to a customised bifunctional version of TCMC (Macrocyclics, Plano, TX, USA), using an enzymatic procedure based on Jeger et al. [16] and Dennler et al. [17], resulting in up to two TCMC molecules conjugated to a specific amino acid in the Fc portion of the antibody. An in vitro stability study in serum up to 72 h at 37 °C demonstrated >96% remains associated with the chelate; this stability is consistent with that reported for 203Pb using the same chelating agent in Chappell et al. [10]. ²¹²Pb was produced using 224Ra generators provided by Orano Med SAS (Bessines-sur-Gartempes, Haute-Vienne, France). Chelation was performed by incubating with 0.1 or 1 mg mAb-TCMC per 40 MBq ²¹²Pb for 15 min at 37 °C in 150 mM ammonium acetate, pH 4.5. Radiochemical purity, assayed by instant thin-layer chromatography, was >94%. All experiments were performed using a specific activity of 37 MBq/mg, except for the early tumour stage RIT experiments, which were performed at both 37 MBq/mg and 370 MBq/mg. The immunoreactivity of ²¹²Pb-anti-CD20 was assessed in vitroby direct binding assays conducted in EL4-hCD20 cells [18], and a KD of 12.6 ± 5.8 nM was obtained, consistent with the known affinity of rituximab (KD = 5.2–11.0 nM) [19]. Furthermore, an immunoreactive fraction >81% was obtained. For single-photon emission computed tomography (SPECT/CT) imaging, mAbs were radiolabeled with 203Pb in 0.5 M HCl (Lantheus Medical Imaging; North Billerica, MA, USA). After adjusting the pH of the 203Pb solution to 4.5 using 1.5 M ammonium acetate, mAbs were incubated for 15 min with 203Pb at 37 °C. The radiochemical purity was >98%, and the specific activity was ~37 MBq/mg.

In vitro studies Cell proliferation assays were performed using Cell Titer Glo (Promega). EL4-hCD20-luc cells were cultured at 37 °C in 96-well plates (25,000 cells in 100 µL/well), and proliferation was assessed on days 1, 2, and 3 after treatment with various concentrations of ²¹²Pb-MAb (0.925, 1.85, 3.7, and 7.4 kBq/mL) or cold Ab (25–200 ng/mL). The number of viable cells was determined (in triplicate) by measuring the absorbance at 490 nm after the addition of a tetrazolium salt for 2 h. Apoptosis was evaluated using Annexin/7AAD dual staining (BD Pharmingen, San Diego, CA, USA) followed by flow cytometry (Accuri C6; BD Biosciences). The proportion of apoptotic cells included early apoptotic cells (Annexin V+/7AAD−), late apoptotic cells (Annexin V+/7AAD+), and necrotic cells (Annexin V−/7AAD+). All in vitro experiments were performed at least three times.

Biodistribution and SPECT/CT imaging Biodistribution experiments were performed in tumour-bearing mice at 25 days after engraftment. ²¹²Pb-rituximab or ²¹²Pb-isotypic control (1.85 MBq) was injected intravenously. At each time point (2, 6, 18, 24, or 48 h post-injection), animals having received ²¹²Pb-rituximab (4–5 animals/group) or ²¹²Pb-isotypic control (2–3 animals/group) were euthanized under isoflurane inhalational anaesthesia by cervical dislocation. BLI was performed 1 day before injection and before sacrifice. Selected tissues were excised and weighed. One femur was counted directly and the other one was extracted and flushed to isolate bone marrow. Tissues radioactivity levels were measured using a calibrated γ-counter (PerkinElmer) with an energy window of 190–290 keV considering ²¹²Pb major emitted gamma (238 keV). Radioactivity uptake in the organs was expressed as the percentage injected dose per gram of tissue (%ID/g) after correcting for radioactive decay at each time point. For SPECT/CT imaging, animals were injected with 203Pb-rituximab (18.5–25 MBq) and imaged under isoflurane inhalational anaesthesia (1.8%, 50% air/50% oxygen, 1.4 L/min) at 6, 24, 48, and 96 h after injection using a MicroSPECT/CT (U-SPECT4/CT; MILabs, Utrecht, The Netherlands) and euthanized at the end of experiment to confirm tumoral presence. Image acquisition lasted 30 min for earlier time points to 90 min for later time points. Energy windows were set over the 279 keV peaks (±20%), and a GP-RM collimator was used (75 holes of 1.5 mm diameter, iterative reconstruction OS-EM, no filter −16 subsets/6 iterations/voxel size: 0.8 mm). The SPECT resolution for 203Pb is estimated to be less than 1 mm [20]. Images were analysed with PMOD Software (PMOD Technologies, Zurich, Switzerland).

Dose range finding and toxicity studies Toxicity studies were conducted in healthy 8-week-old C57BL/6 mice at 7 days, 21 days, or 3 months. Mice received ²¹²Pb-rituximab (185–740 kBq) (7–10/group) by intravenous injection and were compared with mice (5/group) that received PBS, rituximab, isotypic control (100 μg), or 277.5 or 555 kBq 212Pb-isotypic control. Mice were monitored daily and weighed throughout the experiments. Blood was collected at the time of sacrifice (7 days, 21 days, or 3 months). The haemoglobin concentration, white blood cell count, and platelet count were determined using an automated hematology analyzer (Cell Dyn, Abbott Laboratories, Chicago, IL, USA). Biochemical parameters (aspartate amino transferase, alanine amino transferase, urea, and creatinine) were measured in plasma using an automated biochemical analyzer (Konelab, Thermo Fisher Scientific, Waltham, MA, USA).

Radioimmunotherapy studies Treatment was injected 11 days after intravenous injection of EL4-hCD20-luc cells, or once the tumours were detectable by BLI (20–30 days post-injection). After 11 days, mice post-engraftment (early tumour stage) were randomly assigned to experimental groups (16 mice/group) and received a single intravenous injection of ²¹²Pb-rituximab (277.5 kBq) with a specific activity of 370 or 37 kBq/mg, ²¹²Pb-isotypic control (277.5 kBq, 37 kBq/mg), PBS, or rituximab (40 mg/kg). Mice were monitored daily for general clinical signs and were weighed twice a week. Mice were euthanized when they reached their body weight limit (30% below their weight at day 0) or their general status declined, or if tumour progression caused obvious discomfort, as required by the institutional animal care guidelines. Overall survival was measured as an indicator of therapeutic efficacy. For BLI-detectable tumours (late tumour stage), mice were randomly assigned to experimental groups and received a single intravenous injection of ²¹²Pb-rituximab (277.5 kBq, 37 kBq/mg, n = 25), ²¹²Pb-isotypic control (277.5 kBq, 37 kBq/mg, n = 7), PBS (n = 12), or rituximab (40 mg/kg, n = 7), or two injections of ²¹²Pb-rituximab (277.5 kBq, 37 kBq/mg) on days 0 and 7 (n = 9) or days 0 and 14 (n = 11). The general condition of the mice was monitored, and tumour evolution was assessed by BLI before and after treatment.

Statistical methods GraphPad Prism v6 (La Jolla, USA) was used to calculate statistical parameters. All p values are two sided and difference were considered significant for p < 0.05. In-text numbers indicate means of pooled data ± standard deviation (SD) for biodistribution studies and means ± standard error of the mean (SEM) for other studies. For comparison between treatment at the different analysis time (in vitro studies, biodistribution in tumours and blood toxicity studies), two-way analysis of variance were performed. For RIT studies, survival rates were determined by the Kaplan–Meier method and compared using the log-rank test.

Results:

In vitro studies EL4-hCD20-luc cells were treated with increasing activities (0.925–7.4 kBq/mL) of ²¹²Pb-rituximab or ²¹²Pb-isotypic control. Evaluation of cell proliferation for 3 days showed dose-dependent growth inhibition after ²¹²Pb-rituximab treatment (Fig. 1a). From day 1 and at all activities tested, this inhibition was significantly higher than that induced by the ²¹²Pb-isotypic control (p < 0.0001). Similarly, starting at 1.85 kBq/mL, mortality on day 2 was significantly higher (p < 0.01) after ²¹²Pb-rituximab treatment than after ²¹²Pb-isotypic control treatment at the same activity (Fig. 1b). The same experiment with increasing concentrations (25–200 ng/mL) of the two cold mAbs, rituximab and isotypic control, showed no significant effects on growth inhibition or mortality (data not shown).