Targeting both GD2 and B7-H3 using bispecific antibody improves tumor selectivity for GD2-positive tumors.

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 (t 1/2 = 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 [ 89 Zr]Zr-Df-INV34-6, [ 89 Zr]Zr-Df-bsAb CTRL, and [ 89 Zr]Zr-Df-DINU. Importantly, [ 89 Zr]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 [ 89 Zr]Zr-Df-bsAb CTRL control. [ 89 Zr]Zr-Df-DINU displayed similar uptake in both GD2 + tumor models, with uptake comparable to [ 89 Zr]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.