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

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.

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.

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.

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.