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 (t_1/2 = 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 [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, unlike [⁸⁹Zr]Zr-Df-DINU, which displayed elevated tumor uptake, irrespective of hB7-H3 expression. Nontargeted [⁸⁹Zr]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.