双标记抗-GD2靶向探针用于神经母细胞瘤的术中分子成像

手术切除是治疗儿童最常见的颅外实体恶性肿瘤——神经母细胞瘤的关键措施。安全定位和切除原发肿瘤及远处转移灶仍然面临巨大挑战，导致并发症发生率高、手术不完整，影响预后。术中分子成像（IMI）利用靶向放射性或荧光示踪剂在手术中识别和可视化肿瘤。由于GD2在神经母细胞瘤中高度表达且在正常组织中表达极少，选择其作为IMI靶点。通过流式细胞术测定神经母细胞瘤细胞系中的GD2表达。将DTPA和IRDye® 800CW偶联到抗-GD2抗体，制备出DTPA-αGD2-IR800。随后用ELISA测定抗体和非放射性示踪剂的结合亲和力（Kd）。将人源性神经母细胞瘤SK-N-BE(2)细胞注射到3.5-5周龄裸鼠左肾上腺中，构建异种移植肿瘤模型，肿瘤生长5周。通过尾静脉注射111In-αGD2-IR800或同型对照示踪剂。4天和6天后，处死动物，使用伽马计数器和ImageJ分析采集的SPY-PHI荧光影像，测定包括肿瘤、对侧肾上腺、肾脏、肝脏、肌肉、血液等器官的伽马和荧光分布。采用单因素方差分析（对每种示踪剂/天数组合单独分析）比较器官摄取率，若有显著差异，使用Sidak多重比较检验比较各器官与肿瘤的摄取差异。采用手持式设备检测和可视化原位肿瘤，并评估非引导切除后残留病灶。成功合成了含有0.75-2.0 DTPA和2-3 IRDye® 800CW的111In-αGD2-IR800，保持了良好的抗原结合能力（aGD2的Kd=2.39 nM，对照DTPA-aGD2-IR800为21.31 nM）。抗-GD2示踪剂在携带人类神经母细胞瘤异种移植物的动物中表现出特异性摄取（第4天和第6天的肿瘤对血液比率分别为3.87和3.88），而同型对照示踪剂未累积（第4天和第6天分别为0.414和0.514）。利用广泛使用的手术工具（Neoprobe®和SPY-PHI相机）检测并可视化异种移植肿瘤，为手术切除后切除腔中残留病灶的发现提供便利。我们开发了一种双标记抗-GD2抗体示踪剂，结合了In-111和IRDye® 800CW，分别用于放射引导和荧光引导手术。该示踪剂能有效结合GD2，选择性积聚在GD2表达的异种移植肿瘤中，并利用手持近红外相机实现肿瘤的可视化。这些结果支持未来在儿童神经母细胞瘤治疗中应用111In-αGD2-IR800，以提升患者安全性、切除彻底性及整体预后。

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.