O2165: Intraoperative Molecular Imaging of Neuroblastoma with DTPA[In-111]-Dinutuximab-IR800: Optimization to Prepare for Clinical Use

Background: Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of pediatric cancer deaths. Though >90% resection improves outcomes, distinguishing tumor from neurovascular structures and identifying remote disease remains challenging. Intraoperative molecular imaging (IMI) uses targeted tracers to help surgeons identify disease deposits and distinguish tumor from healthy tissues during surgery. Specifically, targeted radioactive isotopes such as indium-111 can be detected through tissue, while targeted fluorophores such as IRDye800CW (IR800) can be visualized with near-infrared fluorescent cameras (NIRC). We designed a dual-labeled IMI tracer, DTPA[In-111]-dinutuximab-IR800, which targets GD2 (overexpressed in 99% of neuroblastoma) for detection of gamma decay and fluorescence visualization. While adequate numbers of DTPA chelators per tracer are established in the literature, here, we modified the number of IR800 fluorophores per antibody (F/Ab) to determine the impact on binding in vitro and accumulation in vivo.

Methods: DTPA-dinutuximab-IR800 was synthesized with approximately 1 DTPA and a range of F/Ab. The fluorescence and binding affinity of each was measured by fluorimeter and ELISA to immobilized GD2. SK-N-BE(2) orthotopic neuroblastoma xenografts were grown in the left adrenal gland of mice. DTPA-dinutuximab-IR800 with different F/Ab were radiolabeled with indium-111 and injected (n=4-6 per group, I.V.). After 4 days, gamma and optical biodistributions were performed using a gamma counter and NIRC. Intraoperative use of the NIRC was also assessed. One or two-way ANOVA was used for statistical comparisons of binding and biodistribution, respectively.

Results: Increasing the number of IR800 per dinutuximab (F/Ab) decreased the binding affinity (Kd 5.2, 18.63, and 75.79 nM for 1.55, 2.70, and 4.37 F/Ab, respectively), though fluorescence per antibody peaked in the middle consistent with quenching (246.4, 379.4, and 327.4 fluorescence units/Ab), and F/Ab did not impact tracer accumulation in neuroblastomas (7.0-10.8 %ID/g for 1.18-2.71 F/Ab; p=ns; Figure 1A). More IR800 per tracer also led to increased non-specific liver accumulation, seen with gamma biodistribution (2.09, 5.55, 7.42, and 8.81 % ID/g tissue; p< 0.0001; Figure 1B) and optical biodistribution (8.6, 10.1, 13.2, and 14.3 fluorescence units, for 1.18, 1.72, 2.14, and 2.71 F/Ab, respectively; p< 0.0001; Figure 1C). Tracer accumulation in tumor was visualized using the handheld SPY-PHI camera both in vivo and ex vivo (Figure 2).

Conclusion: While IR800 substitution levels did not affect tracer accumulation in tumor, increasing the F/Ab decreased GD2 binding affinity (though adequate until at least 2.7 F/Ab), and increased non-specific liver accumulation, potentially impairing the signal-to-noise ratio intraoperatively. Furthermore, the tracer enabled tumor visualization in vivo with the commonly used SPY-PHI intraoperative NIRC. Thus, lower F/Ab enables adequate binding and accumulation while minimizing background signal. With optimization of our tracer, we are poised to focus on clinical translation for use in patients with neuroblastoma.