Anti-cancer immune priming with β-radioligand therapy using a novel high affinity antibody selectively targeting the 4Ig-Isoform of B7-H3

Rationale: Radioligand therapy (RLT) is an emerging oncologic strategy that uses molecularly targeted therapeutic radioisotopes to reduce tumor burden and improve survival in patients with advanced cancers.Expanding RLT to new targets and understanding its systemic immune effects could enhance its clinical impact.B7-H3 (CD276) is an attractive target due to its high differential expression in solid tumors compared to normal tissues.However, the presence of two isoforms, 4Ig-B7-H3 (tumor-associated) and 2Ig-B7-H3 (soluble decoy), poses challenges for selective targeting, especially in the context of RLT.Methods: A novel IgG2a monoclonal antibody (MIL33B) was developed with high affinity for 4Ig-B7-H3 (72 picomolar) and 8-to 18-fold selectivity over soluble 2Ig-B7-H3.Target specificity was assessed using live-cell fluorescence microscopy with AF594-labeled MIL33B.In vivo tumor binding was evaluated by PET-CT imaging with 89 Zr-labeled MIL33B in murine xenograft (HeLa cervical) and syngeneic tumor models (4T1 breast, B16F10 melanoma, CT26 colorectal) expressing human 4Ig-B7-H3.Therapeutic efficacy was tested using 90 Y-labeled MIL33B (100 Ci) in an established CT26 colorectal tumor model.Immunologic effects were analyzed to assess CD8+ T-cell activation and immune memory.Results: MIL33B demonstrated strong membranous localization using live cell fluorescence microscopy.PET-CT imaging with 89 Zr-labeled MIL33B confirmed robust tumor-selective binding in vivo.A single systemic dose of 90 Y-MIL33B achieved 53% long-term survival in a 4Ig-B7-H3-dependent manner in an otherwise fatal CT26 colorectal syngeneic tumor model.Immunologic analysis revealed that 90 Y-MIL33B RLT acted as an immune priming event, engaging CD8+ T-cell activation and inducing immunological memory.Conclusions: MIL33B enables selective targeting of 4Ig-B7-H3 for beta-emitting RLT, overcoming challenges posed by soluble isoforms.These findings support further investigation of MIL33B as a systemic therapeutic with immune-priming potential, either alone or in combination strategies for cancer treatment.