Therapeutic potential of a lead-212 labelled anti-CD146 antibody in mice with mesothelioma

Introduction: Mesothelioma is a rare malignancy that may affect the pleura, peritoneum, pericardium, and the tunica vaginalis. In the peritoneum, it typically presents as a diffuse, extensive metastatic spread throughout the peritoneal cavity. Standard treatment includes surgical resection and systemic chemotherapy. Despite some efficacy of these treatments, most patient subsets face a poor prognosis with a median survival of about one year.

A murine monoclonal antibody (OI-3), against human CD146, has previously been radiolabeled with the beta-emitter lutetium-177 and shown potential as a targeting agent against osteosarcoma. CD146 is a cancer-associated cell surface glycoprotein with high expression in several cancers including mesothelioma, glioblastoma, ovarian and triple negative breast cancer. Here we present proof-of-concept data for targeting of mesothelioma tumors with a lead-212 (²¹²Pb) radiolabeled antibody in vitro and in vivo. Lead-212, a daughter of ²²⁴Ra, serves as an in vivo generator of alpha particles.

Methods: Surface CD146 expression in the MSTO-211H human mesothelioma cell line was measured by flow cytometry using fluorescently tagged antibodies. The OI-3 antibody was conjugated to TCMC and radiolabeled with ²¹²Pb (hereby referred to as ²¹²Pb-OI-3) with quality check performed by evaluating the radiochemical purity (RCP) and immunoreactive fraction (IRF) in a target-positive cell line (OHS). Surface plasmon resonance (SPR) with multi-cycle kinetics was performed on OI-3 with a human CD146 protein for binding evaluation.

The biodistribution study of the ²¹²Pb-OI-3 was performed using age equivalent female athymic nude mice subcutaneously injected with 2.5-5 × 10⁶ MSTO-211H cells on both flanks. Twenty-three days after cell inoculation, mice were intratumorally injected on one selected flank with each mouse receiving either 43 kBq ²¹²Pb-OI-3 or 40 kBq ²¹²Pb-labeled TCMC-conjugated murine IgG isotype (²¹²Pb-mIgG). Mice were sacrificed after 24 h for harvesting and radioactivity quantification of selected organs.

The therapeutic potential of ²¹²Pb-OI-3 was evaluated using age equivalent female athymic nude mice intraperitoneally inoculated with 2.5 × 10⁶ of the MSTO-211H cells. Six days after cell inoculation, mice were randomized into six different groups with each mouse receiving an i.p bolus of either saline, vehicle, non-radiolabeled OI-3, mIgG, 348 kBq ²¹²Pb-OI-3, or 371 kBq ²¹²Pb-mIgG with a constant antibody amount of 10 µg. Mice were followed over the course of the study, monitoring weight every 2-3 days, and sacrificed at predefined humane endpoints corresponding to when the mice displayed significant clinical signs of tumor burden.

Results: Flow cytometry confirmed surface expression of CD146 on the MSTO-211H cell line with histograms showing a good separation from the non-treated control samples with a relative mean fluorescence intensity of 4.4±0.8. RCP of the ²¹²Pb-OI-3 varied from 98-100 %. IRF on target-positive cell line (OHS) varied from 49-69 % specific binding. SPR showed a K_D of 5.1 nM for OI-3 against human CD146.

The biodistribution study showed higher percentage injected radioactivity per gram (% IA/g) of ²¹²Pb in the treated and untreated tumors for ²¹²Pb-OI-3 with mean values 35.4 and 7.8% IA/g, respectively, compared to 20.7 and 5.3% IA/g for the ²¹²Pb-mIgG. However, the difference was not statistically significant in neither tumor nor any other tissues when comparing the ²¹²Pb-OI-3 vs ²¹²Pb-mIgG.

Mice treated with ²¹²Pb-OI-3 showed a significantly prolonged survival time compared to the saline, vehicle and non-labeled OI-3 controls. The median survival time of the ²¹²Pb-OI-3 group was 55 days, while the median survival for saline, vehicle, non-labelled OI-3 and ²¹²Pb-mIgG was 40, 40, 41, 39 and 42 respectively.

Conclusions: This lead-212-labeled anti-CD146 antibody represents a promising targeted radiotherapeutic option against mesothelioma.