乳腺癌前哨淋巴结活检术新型荧光靶向示踪剂的基础与应用研究

Sentinel lymph node biopsy (SLNB) has become the standard procedure for the axillary stage in early breast cancer patients, with the combining use of radioisotope and blue dye. However, the extensive application of SLNB has been restricted by the poor specificity and access issue of the traditional tracer. The fluorescence image of axillary lymph node can be obtained through the guidance of the indocyanine green. Unfortunately, it has the shortcoming of the second level lymph node imaging. Rituximab is a lymph targeted drug and is a human specific monoclonal antibody against the CD20 molecule in the B type lymphocyte membrane. In this study, a novel sentinel lymph node tracer with the advantages of both drugs was produced by the coupling of them. The characteristics in vitro of the novel tracer, such as the labeling rate, the immune activity and the molecular integrity will be made clear though the first step study. After the safety limitation test, the best dosage, the sustained localization effect and the optimal time window of the tracer will be investigated, using the hind limb drainage in mice as an animal model. Then the success rate and the accuracy of SLNB with the guide of the novel tracer will be evaluated by the clinical research. Meanwhile, the optimal dose, the optimal time of injection, the adverse reactions and the threshold value will also be explored. The purpose of our study is to clarify the biological characteristics of the novel fluorescent-targeted tracer, and to establish the technology system of the SLNB procedure with the guidance of it. Accordingly, the foundation for the clinical application of the novel tracer will be set through the established result of the study.

前哨淋巴结活检术是评估乳腺癌腋窝淋巴结状况的微创诊断技术，由于传统示踪剂定位特异性差及药物准入等问题，限制了其在临床的推广应用。荧光示踪剂吲哚菁绿通过荧光成像可获得腋窝淋巴结的荧光图像，但具有使次级淋巴结显像的缺点。利妥昔单抗是一种针对淋巴结中B淋巴细胞膜上CD20分子的特异性人源化单克隆抗体，课题组前期将吲哚菁绿偶联于利妥昔单抗作为新型示踪剂，将兼顾荧光显像与靶向定位的优点。本项目将通过检测新型示踪剂的标记率、免疫活性和分子完整性以明确其生物特性，建立动物模型进行安全性、最佳注射剂量、持续定位效应和最佳显像时间窗研究，随后通过人体验证研究评估新型示踪剂进行前哨淋巴结活检术的成功率和准确性、人体最佳剂量、最佳注射时间、检测阈值和不良反应。目的在于明确新型荧光靶向示踪剂的生物学特性，建立其应用于前哨淋巴结活检术的技术体系，为新型荧光靶向示踪剂引导的乳腺癌前哨淋巴结活检技术的广泛应用奠定基础。

前哨淋巴结活检术是评估早期乳腺癌腋窝淋巴结状况的微创诊断技术，由于传统示踪剂定位特异性差及药物准入等问题，限制了其在临床上的推广应用。传统荧光示踪剂吲哚菁绿通过荧光成像可获得腋窝淋巴结的荧光图像，但具有使次级淋巴结显像的缺点。利妥昔单抗是一种针对淋巴结中B淋巴细胞膜上CD20分子的特异性人源化单克隆抗体。本研究通过直接偶联法制备前哨淋巴结新型荧光靶向示踪剂吲哚菁绿-利妥昔单抗，双层析快速薄层层析-硅胶层析纸法测定利妥昔单抗大分子上吲哚菁绿的标记率为100%，非还原型SDS聚丙烯酰胺凝胶电泳法和双抗体夹心间接酶联免疫测定法检测新型示踪剂中利妥昔单抗分子完整且保持了单抗的免疫活性，按中华药典要求检测新型示踪剂为无菌、无致热原的溶液且局部注射不会产生危害，在前哨淋巴结动物模型上能够准确定位到前哨淋巴结，荧光显像效果最佳的利妥昔单抗与吲哚菁绿质量比例为4: 1、6: 1，临床验证研究证实与放射性同位素联合蓝染料法的金标准对比，吲哚菁绿-利妥昔单抗前哨淋巴结活检的符合率为86.2%（258/290），准确度为99.6%（257/258），敏感性为96.7%（33/34），特异度为100%（225/225），假阴性率为2.9%（1/34），随着新型荧光靶向示踪剂剂量减少，各组前哨淋巴结的荧光显影率也依次降低。吲哚菁绿-利妥昔单抗偶联的前哨淋巴结新型荧光靶向示踪剂的制备工艺简单且无放射性危害，其中单抗的分子完整性和免疫活性无破坏，为无菌、无致热原、无急性毒性的示踪剂，能够用于前哨淋巴结的靶向定位显像，为乳腺癌前哨淋巴结活检技术的广泛应用奠定了基础。