双标记的抗GD2靶向探针，用于神经母细胞瘤的术中分子成像

手术切除是治疗神经细胞瘤的组成部分，这是儿童最常见的颅外固体恶性肿瘤。安全定位和恢复原发性肿瘤和疾病的远程沉积仍然是一个重大挑战，导致并发症和不完整的手术发生率，结果恶化。术中分子成像（IMI）使用靶向放射性或荧光示踪剂来识别和可视化术中肿瘤。选择GD2作为IMI靶标，因为它在神经母细胞瘤中高度过表达并在正常组织中最小表达。通过流式细胞仪测量神经母细胞瘤细胞系中的GD2表达。 DTPA和IRDYE®800CW与抗GD2抗体共轭以生成DTPA-αGD2-IR800。然后通过ELISA测定法测量抗体和非二醇标记示踪剂的结合亲和力（KD）。将人类神经母细胞瘤SK-N-BE（2）细胞手术注射到3.5-5周龄大的裸鼠的左肾上腺中，而原位异种移植肿瘤持续了5周。通过尾静脉注射对111IN-αGD2-IR800或同种型对照示踪剂进行施用。 4天和6天后，将小鼠安乐死，并使用伽马计数器进行伽马和荧光生物分布，并对所获得的器官的间谍荧光图像进行imageJ分析（包括肿瘤，对侧肾上腺，肾脏，肾脏，肝脏，肌肉，血液等）。通过单向方差分析比较器官的摄取（对每种示踪剂/天组合进行单独的分析），如果显着，Sidak的多重比较测试将使用以比较每个器官对肿瘤的摄取。手持式工具还用于检测和可视化肿瘤原位，并在非引导切除后评估残留疾病。111IN-αGD2-IR800与0.75-2.0 DTPA成功合成，每个抗体和2-300cw的每抗体和抗体抗体抗原抗原结合（kd = 2.39 nm = 2.39 nm）nm = 2.39 nm。 DTPA-AGD2-IR800）。抗GD2示踪剂在人类神经母细胞瘤异种移植物（γ生物分布肿瘤与血统比率为3.87和3.88的小鼠中表现出抗原特异性摄取，使用抗GD2截止器为3.87和3.88），而同型对照器则不累积（0.414和0.414和0.514和6和6和6和6和6和60）。 Probe accumulation in xenografts was detected and visualized using widely available operative tools (Neoprobe® and SPY-PHI camera) and facilitated detection ofputative residual disease in the resection cavity following unguided resection.We have developed a dual-labeled anti-GD2 antibody-based tracer that incorporates In-111 and IRDye® 800CW for radio- and fluorescence-guided surgery, respectively.示踪剂与GD2充分结合，特别积累了表达GD2的异种移植肿瘤，并使用手持式NIR摄像头实现肿瘤可视化。这些结果鼓励开发111IN-αGD2-IR800，用于神经母细胞瘤儿童的未来使用，目的是提高患者的安全性，切除的完整性和整体患者结果。

Surgical resection is integral for the treatment of neuroblastoma, the most common extracranial solid malignancy in children. Safely locating and resecting primary tumor and remote deposits of disease remains a significant challenge, resulting in high rates of complications and incomplete surgery, worsening outcomes. Intraoperative molecular imaging (IMI) uses targeted radioactive or fluorescent tracers to identify and visualize tumors intraoperatively. GD2 was selected as an IMI target, as it is highly overexpressed in neuroblastoma and minimally expressed in normal tissue.GD2 expression in neuroblastoma cell lines was measured by flow cytometry. DTPA and IRDye® 800CW were conjugated to anti-GD2 antibody to generate DTPA-αGD2-IR800. Binding affinity (Kd) of the antibody and the non-radiolabeled tracer were then measured by ELISA assay. Human neuroblastoma SK-N-BE(2) cells were surgically injected into the left adrenal gland of 3.5-5-week-old nude mice and the orthotopic xenograft tumors grew for 5 weeks. 111In-αGD2-IR800 or isotype control tracer was administered via tail vein injection. After 4 and 6 days, mice were euthanized and gamma and fluorescence biodistributions were measured using a gamma counter and ImageJ analysis of acquired SPY-PHI fluorescence images of resected organs (including tumor, contralateral adrenal, kidneys, liver, muscle, blood, and others). Organ uptake was compared by one-way ANOVA (with a separate analysis for each tracer/day combination), and if significant, Sidak's multiple comparison test was used to compare the uptake of each organ to the tumor. Handheld tools were also used to detect and visualize tumor in situ, and to assess for residual disease following non-guided resection.111In-αGD2-IR800 was successfully synthesized with 0.75-2.0 DTPA and 2-3 IRDye® 800CW per antibody and retained adequate antigen-binding (Kd = 2.39 nM for aGD2 vs. 21.31 nM for DTPA-aGD2-IR800). The anti-GD2 tracer demonstrated antigen-specific uptake in mice with human neuroblastoma xenografts (gamma biodistribution tumor-to-blood ratios of 3.87 and 3.88 on days 4 and 6 with anti-GD2 tracer), while isotype control tracer did not accumulate (0.414 and 0.514 on days 4 and 6). Probe accumulation in xenografts was detected and visualized using widely available operative tools (Neoprobe® and SPY-PHI camera) and facilitated detection ofputative residual disease in the resection cavity following unguided resection.We have developed a dual-labeled anti-GD2 antibody-based tracer that incorporates In-111 and IRDye® 800CW for radio- and fluorescence-guided surgery, respectively. The tracer adequately binds to GD2, specifically accumulates in GD2-expressing xenograft tumors, and enables tumor visualization with a hand-held NIR camera. These results encourage the development of 111In-αGD2-IR800 for future use in children with neuroblastoma, with the goal of improving patient safety, completeness of resection, and overall patient outcomes.