高亲和力αVβ3 和TNF双受体靶向乳腺癌正电子成像探针的构建

The stage of breast cancer is criticl for its clinical prognosis.Unfortunately, at present the imaging modality that possesses both high sensitivity and specificity in the early detection of breast cancer is not available. The RGD peptide, although being used for noninvasive imaging in a variety of integrin positive tumors, dosen't show an ideal sensitivity for early-detection of the breast cancer in our ongoing clinical research. Supported by the NSFC, we have developed a TNF receptor tagetting octapeptide Ala-Thr-Ala-Gln-Ser-Ala-Tyr-Gly (WH701). In this study, we are going to design a dual αvβ3 and TNFR targeted heterodimeric peptide, RGD-WH701, which would contain both c(RGDyK) and WH701 motifs in one single molecule. We intend to determine whether the dual-targeted heterodimer would exhibit significantly improved breast cancer-targeting efficiency both in vitro and in vivo compared with 18F-labeled RGD or WH701 monomers.Through this investigation,we will develop a novel heterodimeric probe which targets breast cancer with high binding affinity and establish a promising PET/CT imaging modality for diagnosing breast cancer in the early stage. The synergistic effects of intergrin and TNF receptor tagetting heterodimer may also encourage further investigations of developing other molecular probes for other toumors with dual or multiple-receptor targeting strategy.

乳腺癌诊断的早晚其预后有天壤之别，但目前临床缺乏早期诊断乳腺癌灵敏性和特异性俱佳的成像手段。业已证实乳腺癌高表达整合素受体αVβ3和肿瘤坏死因子（TNF）受体。申请人在前期应用靶向αVβ3的RGD多肽核素成像诊断乳腺癌的临床研究中发现其灵敏性尚不理想。在NSFC资助下申请人曾筛选出亲肿瘤的靶向TNF受体多肽WH-701。我们拟合成基于RGD和WH701结构的融合肽，并进行F-18标记制备双靶点正电子成像探针F-18-RGD-WH701。通过体内外实验，论证该探针较之单肽探针F-18-RGD和F-18-WH701对于乳腺癌具有更好的靶向性能。 本项目不仅可获得一种新型高亲和力靶向乳腺癌的分子成像探针、从而为乳腺癌早期PET/CT诊断新技术的临床转化创造条件，而且可能为乳腺癌的核素靶向内照射治疗药物的研发建立基础。本研究的策略也可为其他αVβ3 和TNF受体高表达实体肿瘤的相关研究提供借鉴。

乳腺癌的早期检出和诊断对其预后直关重要，但目前临床缺乏早期诊断乳腺癌灵敏性和特异性俱佳的成像手段。本项目在申请人前期相关工作的基础上，合成了基于靶向整合素受体的RGD和靶向肿瘤坏死因子受体的WH701 结构的双靶点融合肽，并进行F-18标记制备双靶点正电子成像探针[18F]-NOTA-Gly3-E（2PEG4-RGD-WH701），利用MCF7和MDA-MB-231两种乳腺癌细胞系，通过体内外实验，对双靶点探针的靶向性进行验证。在注射探针40min时PET扫描结果显示，MCF7移植肿瘤对融合肽的摄取值（%ID/g）为0.93±0.11，而对单肽探针[18F]-NOTA-RGD、[18F]-NOTA-WH701的摄取值为0.76±0.18, 0.44±0.08；MDA-MB231肿瘤对融合肽的摄取值为0.87±0.14，而对单肽探针[18F]-NOTA-RGD、[18F]-NOTA-WH701 的摄取值为0.73±0.13, 0.71±0.28。相比于单肽探针，融合肽在MCF7肿瘤中摄取绝对值和靶/非靶比值都是最高。通过共注射冷肽RGD-WH701，MCF7和MDA-MB-231肿瘤对融合肽的摄取分别降低为0.31±0.02和0.21±0.02，证实融合肽是特异性靶向肿瘤的αvβ3和TNFR1受体。.另外，本项目利用白蛋白结合基团Evans Blue配合物修饰的RGD多肽，构建了长循环多肽PET探针。前期实验结果显示，靶向分子NOTA-EB-RGD在保留了EB对白蛋白的亲和力的同时，也保留了RGD肽对整合素αvβ3的特异性结合能力。PET显像结果显示，注射[64Cu]NOTA-EB-RGD 24 h后的肿瘤摄取率高达16.64 ±1.99 %ID/g，为[64Cu]NOTA-RGD单体肽的近17倍。.本项目研究工作达到了预期的目标，成功合成了具有靶向整合素受体和TNF受体的双靶向融合肽正电子成像探针，细胞学和荷瘤动物实验初步结果显示新型探针可提高乳腺癌的特异性摄取，从而可能提高对于乳腺癌病灶探测的灵敏性；为进一步诊疗一体化放射性药物的研发打下了基础。不过在不同的细胞模型结果存在差异，探针的的优化还有待进一步深入研究。同时，本项目资助下，我们开展了在延长多肽探针的循环时间以提高肿瘤对于探针摄取探索了新的策略。在本项目资助下发表论文七篇，申请专利一项。