ImmunoPET Demonstrates that Co-Targeting GD2 and B7-H3 with Bispecific Antibodies Enhances Tumor Selectivity in Preclinical Melanoma Models.

Disialoganglioside 2 (GD2) is overexpressed in multiple cancers, such as melanoma and neuroblastoma, but also in peripheral nerves. To improve current GD2-targeting approaches, next-generation heterodimeric bispecific human IgG antibodies were created, each with one antibody binding fragment (Fab) arm specific for GD2 and the other Fab arm specific for B7-H3 (CD276) to drive tumor selectivity. The avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) were determined by flow cytometry and competition binding assays in GD2/hB7-H3 B78 cells. INV34-6 showed high avidity for GD2/hB7-H3 but not GD2/hB7-H3 B78 cells, contrasting with the similar cell binding to these cells observed with the anti-GD2 antibody Dinutuximab (DINU). The bispecific antibodies, DINU, and a nontargeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 ( = 78.4 h). Positron emission tomography (PET) corroborated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU in GD2/hB7-H3 and GD2/hB7-H3 B78 tumor models. PET in mice bearing the GD2/hB7-H3 and GD2/hB7-H3 B78 murine xenografts showed similar biodistribution in normal tissues for [Zr]Zr-Df-INV34-6, [Zr]Zr-Df-bsAb CTRL, and [Zr]Zr-Df-DINU. Importantly, [Zr]Zr-Df-INV34-6 tumor uptake was selective to GD2/hB7-H3 B78 over GD2/hB7-H3 B78 tumors, unlike [Zr]Zr-Df-DINU, which displayed elevated tumor uptake, irrespective of hB7-H3 expression. Nontargeted [Zr]Zr-Df-bsAb CTRL isotype control showed markedly lower uptake in all tested tumor models. Overall, bispecific antibodies binding GD2 and B7-H3 showed improved selectivity for targeting tumor cells expressing both antigens. This approach may enhance antitumor efficacy while addressing the toxicity limitations of current GD2-targeting therapies by reducing off-tumor GD2 binding in nerves.