新型放射免疫治疗药物X-HOPO-mAb的研究

The purpose of the research project is to develop a special radioimmunoconjugate labeled with radionuclide Lu -177 or Y-90 for radioimmunotherapy (RIT) of solid tumors overexpressing EGFR. The modified Nimotuzumab is a humanized monoclonal antibody (mAb) secreted by CHO, which recognizes the external domain of the epidermal growth factor receptor (EGFR) with high specificity. A new powerful chelater carboxyl linker-HOPO(hydroxypyridinone) will be synthesized and identified with Mass Spectrometer and NMR, it is coupled to a peptide with the modified Nimotuzumab by dehydration condensation reaction using condensing enzyme. The high specificity and affinity of the obtained complex compound (linker-HOPO-mAb) will be maintained when incubated with human squamous carcinoma cell line A431 overexpressing EGFR , meanwhile its chelating ability to radionuclide Lu -177 or Y-90 will be unchanged. The simple "one step" labeling method of the linker-HOPO-mAb labeled with DTPA-Y90 or carrier-free Lu -177 will be developed. The radiochemical purity and labeling efficiency of the labeled conjugates (X- - HOPO-mAb ) will be determined by thin-layer chromatography, the biological activities of the X- - HOPO-mAb will be measured by competitive inhibition experiments against free nimotuzumab. The pharmacokinetics of the X- - HOPO-mAb in health mice will be analysed, and the biodistribution in healthy mice or bearing tumor xenograft mice will be estimated. The tumor uptake and the absorbed dose of X- - HOPO-mAb will be calculated. The therapeutic effect of the X- - HOPO-mAb will be observed among the bearing tumor xenografts compared with free nimotuzumab. The results of the research will be benefit to develop X-HOPO-mAb as a potential targeting radiopharmaceutical for RIT of tumors overexpressing EGFR with characteristics of powerful effect, low toxicity, easy labeling and more stability.

本课题设想以EGFR为靶点，研发一种标记放射性核素177Lu 或90 Y用于治疗EGFR表达阳性实体瘤的新型放射免疫治疗药物。以改进后的低免疫原性尼妥珠单抗为载体， 设计、合成新型螯合剂linker-HOPO, 并用质谱、核磁共振谱鉴定。通过酶催化其分子一端的羧基与抗体分子上的氨基进行肽缩合反应，得到具有极高螯合能力和亲和力的改进型尼妥珠单抗linker-HOPO，研究一步法标记放射性核素90Y（有载体）、177Lu，获得在体内稳定性高、亲和力强的RIT药物X-DOPO-mAb。通过薄层层析方法测定标记效率和放化纯度，竞争结合实验测定其生物活性。通过动物实验，了解其在正常小鼠体内的分布、代谢规律，评价其对荷瘤鼠的抑瘤和杀伤肿瘤的治疗效果，计算在荷瘤鼠中肿瘤和其它组织器官中的剂量沉积，为开发新型高效、低毒、易标记且稳定性高的新型靶向EGER的RIT药物提供实验和理论依据。

本课题研究的目的是研发一种能够与放射性核素Lu -177 or Y-90有高度螯合能力的、同时又与EGFR具有高度亲和力和特异性结合的肿瘤靶向治疗药物。首先设计与合成了两种HOPO（羟基吡啶酮衍生物），分别为Di-HOPO- 马来酰胺和四齿HOPO，并用质谱和核磁共振谱进行鉴定。螯合能力测定发现四齿HOPO与90Y3+ 具有更好地螯合能力。第二是设计并合成了四条与EGFR有高度特异性结合的多肽，并通过酰化缩合反应与四齿HOPO结合，获得四种连接分子，分别与KB细胞孵育30分钟，激光共聚焦显微镜发现HOPO2-SPX-2 和 SPW127121具有更好的细胞亲和性。 第三是建立了基于两种肿瘤细胞系的四种荷瘤鼠模型，发现当转染并高表达SPATA5L1后，肿瘤生长加快，但局部和肝脏转移被抑制，荷瘤鼠的生存率显著增加（p<0.05)。第四是进行了放射性治疗实验，在经过9 Gy照射治疗后，单纯电离辐射治疗，对具有高度迁移和侵袭能力的肿瘤细胞KB模型的治疗效果并不理想，而结合转染SPATA5L1基因并高表达蛋白，能够显著提高放射治疗效果、抑制肿瘤的生长，控制局部和远端脏器如肝脏的转移，提高生存率。第五是基于新合成的HOPO与重金属离子的螯合特性，研发了一种放射性介入诊疗的辐射防护手套，已经获得国家二类医疗器械注册证书。

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