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.

Fig. 1. In vitro studies.

Percentage of growth inhibition (a) and mortality (b) for increasing ²¹²Pb-mAb activity (0.925–7.4 kBq/mL). a EL4-hCD20-luc cell growth inhibition percent was calculated, from days 1 to 3, using untreated cells as controls on the same day. Data are expressed as the mean ± SEM percentage of growth inhibition ((1 – OD_treatment /OD_control) * 100). b Mortality was evaluated on day 2 by flow cytometry using Annexin V/7AAD dual staining to evaluate early apoptotic, late apoptotic, and necrotic cells.

Fig. 2. Biodistribution and SPECT-CT imaging.

Biodistribution of ²¹²Pb-rituximab (a) and the ²¹²Pb-isotypic control (b) in tumour-bearing mice. Mice were injected with 185 kBq ²¹²Pb-mAbs and then sacrificed at 2, 6, 18, 24, or 48 h post-injection. Radioactivity in tumours, organs, and blood was expressed as the mean ± SD percentage injected dose per gram of tissue (%ID/g). In tumours, two-way analysis of variance showed a significant difference between the two mAbs (p < 0.006). In vivo imaging of ²⁰³Pb-rituximab in tumour-bearing mice (c) and healthy mice (d). ²⁰³Pb-rituximab at 18.5–25 MBq was injected. Mice were imaged by MicroSPECT/CT at 6, 24, 48, and 96 h after injection. For mice bearing tumours (25 days after engraftment), BLI was performed before injection of ²⁰³Pb-rituximab and after the last MicroSPECT/CT acquisition. Each line represents the same mouse imaged at different times. Tumours are indicated by different arrows, allowing monitoring of tumour uptake at the same site. Tumoral presence was confirmed post-mortem at the end of experiment.

Fig. 3. Toxicity studies.

A Effect of ²¹²Pb-rituximab on the survival of healthy mice. Three time points were evaluated: 7 days, 21 days, and 3 months post-injection. Values in bold indicate when lethality occurred. B ²¹²Pb-rituximab toxicity studies based on haematological parameters. The white blood cell count (a), platelet count (b), and haemoglobin concentration (c) were measured at 7 days, 21 days, and 3 months post-injection of PBS, ²¹²Pb-isotypic control (277.5 or 555 kBq), or ²¹²Pb-rituximab (185–740 kBq). Data represent the mean ± SEM. *All mice died before the endpoint.

Fig. 4. Efficacy of ²¹²Pb-rituximab treatment in a murine syngeneic lymphoma model at an early stage.

At 11 days post-engraftment, mice received the following treatments (n = 16 per group): PBS, 40 mg/kg rituximab, 277.5 kBq ²¹²Pb-isotypic control, and 277 kBq ²¹²Pb-rituximab with a specific activity of 370 MBq/mg or 37 MBq/mg. Survival rates were determined by the Kaplan–Meier method and compared using the log-rank test. All treatments except the ²¹²Pb-isotypic control induced a significant increase in MST compared with PBS.

Fig. 5. Efficacy of ²¹²Pb-rituximab treatment in a late-stage murine syngeneic lymphoma model.

Once the tumour was detected by BLI (20–30 days after cell injection), mice were treated with an injection of PBS, rituximab (40 mg/kg), ²¹²Pb-isotypic control (277.5 kBq), ²¹²Pb-rituximab (277.5 kBq), or two injections of ²¹²Pb-rituximab (277.5 kBq) on days 0 and 7 or days 0 and 14. Survival rates were determined by the Kaplan–Meier method and compared using the log-rank test. All ²¹²Pb-rituximab treatments induced a significant increase in the MST compared with PBS (p ≤ 0.001).

Fig. 6. BLI monitoring in a late-stage murine syngeneic lymphoma model.

When the tumour was detected by BLI (20–30 days after cell injection), mice were injected with PBS, rituximab (40 mg/kg), ²¹²Pb-isotypic control (277.5 kBq), ²¹²Pb- rituximab (277.5 kBq) on day 0 (T0), or ²¹²Pb-rituximab (277.5 kBq) on T0 and day 7 (T7) or T0 and day 14 (T14). Mice were monitored by BLI at the indicated time points to follow tumour evolution until sacrifice or the end of the experiment.