RESUMO
Objectives: Disialoganglioside 2 (GD2), overexpressed by cancers such as melanoma and neuroblastoma, is a tumor antigen for targeted therapy. The delivery of conventional IgG antibody technologies targeting GD2 is limited clinically by its co-expression on nerves that contributes to toxicity presenting as severe neuropathic pain. To improve the tumor selectivity of current GD2-targeting approaches, a next-generation bispecific antibody targeting GD2 and B7-H3 (CD276) was generated. Methods: Differential expression of human B7-H3 (hB7-H3) was transduced into GD2+ B78 murine melanoma cells and confirmed by flow cytometry. We assessed the avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) towards GD2+/hB7-H3- B78 cells relative to GD2+/hB7-H3+ B78 cells using flow cytometry and competition binding assays, comparing results an anti-GD2 antibody (dinutuximab, DINU). The bispecific antibodies, DINU, and a non-targeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 (t1/2 = 78.4 h). Using positron emission tomography (PET) studies, we evaluated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU using GD2+/hB7-H3+ and GD2+/hB7-H3- B78 tumor models. Results: Flow cytometry and competition binding assays showed that INV34-6 bound with high avidity to GD2+/hB7-H3+ B78 cells with high avidity but not GD2+/hB7-H3+ B78 cells. In comparison, no selectivity between cell types was observed for DINU. PET in mice bearing the GD2+/hB7-H3- and GD2+/hB7-H3+ B78 murine tumor showed similar biodistribution in normal tissues for [89Zr]Zr-Df-INV34-6, [89Zr]Zr-Df-bsAb CTRL, and [89Zr]Zr-Df-DINU. Importantly, [89Zr]Zr-Df-INV34-6 tumor uptake was selective to GD2+/hB7-H3+ B78 over GD2+/hB7-H3- B78 tumors, and substantially higher to GD2+/hB7-H3+ B78 than the non-targeted [89Zr]Zr-Df-bsAb CTRL control. [89Zr]Zr-Df-DINU displayed similar uptake in both GD2+ tumor models, with uptake comparable to [89Zr]Zr-Df-INV34-6 in the GD2+/hB7-H3+ B78 model. Conclusion: The GD2-B7-H3 targeting bispecific antibodies successfully improved selectivity to cells expressing both antigens. This approach should address the severe toxicities associated with GD2-targeting therapies by reducing off-tumor GD2 binding in nerves. Continued improvements in bispecific antibody technologies will continue to transform the therapeutic biologics landscape.
RESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease resulting in irreversible scarring within the lungs. However, the lack of biomarkers that enable real-time assessment of disease activity remains a challenge in providing efficient clinical decision-making and optimal patient care in IPF. Fibronectin (FN) is highly expressed in fibroblastic foci of the IPF lung where active extracellular matrix (ECM) deposition occurs. Functional upstream domain (FUD) tightly binds the N-terminal 70-kilodalton domain of FN that is crucial for FN assembly. In this study, we first demonstrate the capacity of PEGylated FUD (PEG-FUD) to target FN deposition in human IPF tissue ex vivo. We subsequently radiolabeled PEG-FUD with 64Cu and monitored its spatiotemporal biodistribution via µPET/CT imaging in mice using the bleomycin-induced model of pulmonary injury and fibrosis. We demonstrated [64Cu]Cu-PEG-FUD uptake 3 and 11 days following bleomycin treatment, suggesting that radiolabeled PEG-FUD holds promise as an imaging probe in aiding the assessment of fibrotic lung disease activity.