Molecular Imaging and Targeted Therapy of HER2-Positive Breast Cancers

背景与意义HER2受体在乳腺癌中的表达与预后不良相关，其在远处转移灶中的表达可能与原发灶不同。该项目将为评估乳腺癌（包括转移）中HER2的全球表达提供手段，并为HER2阳性细胞提供特异性治疗药物。作为靶向剂，我们建议使用从瑞典CRADA合作伙伴获得的affbody分子（http://www.affibody.com）。这些非常稳定和高可溶性的螺旋蛋白相对较小（8.3 kDa），可以很容易地在细菌系统中表达或通过肽合成产生。His6-Zher2:324以高亲和力（22 pM）与HER2受体结合，并在羧基末端与半胱氨酸结合以促进结合。为了成像，这些分子可以用放射性核素标记。在治疗方面，His6-Zher2:324将与热敏脂质体偶联，用体内成像的beacon标记，并装载治疗剂（例如毒素，放射增敏剂或激酶抑制剂），将允许通过实时监测其分布来定义局部药物释放。尽管多次尝试将脂质体用于癌症治疗的亲水性和疏水性药物的定点递送，但由于对脂质体与细胞相互作用的了解不足以及各种配方的次优生物分布概况，它们的应用受到限制。我们将使用具有靶向、成像和最佳药物释放能力的多功能脂质体来规避这些问题。我们的策略，包括评估单个患者的靶标存在和分布，然后优化，针对靶标的药物递送，可能显著提高乳腺癌治疗的疗效，同时减少副作用。用显像剂标记。使用马来酰亚胺化学将附着体分子与AlexaFluor或18F偶联，分别用于光学和PET成像。具有最佳脂质组成的脂质体，对37℃以上的温度敏感。脂质体在37℃-45℃温度范围内的热不稳定性将通过荧光光谱法监测钙黄蛋白泄漏来测定。下一步将包括优化技术，通过马来酰亚胺基团将her2特异性附着体分子偶联到脂质体表面，并用光学、PET和MRI显像剂进行标记。由于脂质体的大小是其生物分布的重要决定因素，我们将利用纳米技术表征实验室的资源表征所得到的偶联物的大小。脂质体将根据已发表的方法装载治疗剂。我们将向CCR的临床肿瘤学家咨询治疗乳腺癌的最佳药物。利用结合、增殖和克隆生存试验以及分子生物学方法，我们将在体外彻底表征缀合物的结合特性及其对靶细胞的影响。我们还将研究它们结合对受体表达和治疗性抗体（赫赛汀）结合的影响。结合物的生物分布将在携带her2阳性肿瘤异种移植物的裸鼠身上进行研究。为了测试放射性缀合物的体内成像能力，我们将通过光学或PET成像监测Herceptin或DMAG治疗后肿瘤异种移植物中HER2的预期下调。从影像学数据中获得的表达水平估计将通过免疫组织化学和Western blots对肿瘤组织进行离体分析来验证。我们的药物输送系统的初步体内研究将使用装载亲疏水荧光标记的脂质体进行，允许绘制和跟踪脂质体在体内的组织/器官区隔，并监测药物在37℃-45℃的泄漏和/或释放。将优化基于聚焦超声技术的非侵入性方法来分解脂质体。MRI和微pet可用于优化这些模式的共轭物的成像能力。我们将对her2阳性荷瘤动物进行实验治疗，以评估肿瘤靶向性热敏脂质体递送治疗剂与目前的应用方法相比，疗效的提高。对于探索性IND研究，可以使用单一哺乳动物物种来建立安全边际。开发了光学成像修饰物-AlexaFluor偶联物的项目状态探针，并在体外和体内与赫赛汀-AlexaFluor偶联物进行了比较。我们在体外证明，与赫赛汀相比，附着体分子与HER2的不同表集结合，并且与赫赛汀相比，附着体对HER2信号通路的影响有限。AlexaFluor标记的her2 - cys粘附体和赫赛汀在人乳腺癌和卵巢癌异种移植裸鼠体内进行了比较成像研究。虽然观察到heeptin的高肿瘤特异性积累，但在肿瘤处只能检测到微弱的信号，这很可能是由于其从血液中快速清除和alexafluor标记的ZHER2-Cys粘附体在肾脏中的高积累。为了增加粘附体分子在血液中的时间，含有白蛋白结合域的分子已被AlexaFluor标记。对这些偶联物进行了生物分布研究。用于贴体分子18F标记的PET成像探针正在开发中。体外研究结果表明，与未标记的词缀分子预先孵育以剂量依赖的方式抑制放射性偶联物与细胞的结合。His6-ZHer2:342-Cys-18F与细胞膜结合的饱和分析显示只有一类高亲和力的结合位点。体内PET显像显示，早在注射后20分钟肿瘤内放射性就开始高积累，60分钟后未达到平台期。生物分布研究结果证实了这一点，即注射后1小时，肿瘤中的放射性浓度（%ID/g）比血液中的放射性浓度高约10倍。三小时后，这一比例上升至近17。相反，肾脏中最初的高浓度（与肿瘤中的浓度相当）同时下降了三倍。体外表征了热敏脂质体与her2特异性、her2 - cys粘附体分子的偶联物。我们研究了her2阳性SKBR-3细胞和her2阴性U251细胞中修饰脂质体与未修饰脂质体摄取的时间依赖性。在22℃和37℃两种温度下，脂质体结合无显著差异。经过30分钟的孵育，我们发现her -2阳性细胞中修饰的脂质体比未修饰的脂质体富集8倍。随着附着物浓度的降低，我们观察到非特异性条带明显降低。未修饰脂质体孵育2h后的细胞荧光与未处理的对照组相当[摘要截断为7800个字符]

Background and Significance Expression of HER2 receptors in breast cancers is correlated with poor prognosis and their expression may be different in distant metastases as compared to the primary tumor. This project will provide means to assess global expression of HER2 in breast cancers (including metastases) and to deliver therapeutic agents specifically to HER2-positve cells. As the targeting agent we propose to use Affibody molecules obtained from our CRADA partner in Sweden (http://www.affibody.com). These very stable and highly soluble α-helical proteins are relatively small (8.3 kDa) and can be readily expressed in bacterial systems or produced by peptide synthesis. The His6-Zher2:324 binds to HER2 receptors with high affinity (22 pM) and is available with cystein at the carboxy-terminal to facilitate conjugation. For imaging purposes, these molecules with be labeled with radionuclides. For therapy, the His6-Zher2:324 will be conjugated with thermo-sensitive liposomes that, labeled with beacons for in vivo imaging and loaded with therapeutic agents (e.g. toxins, radiosensitizers or kinase inhibitors), will allow local drug release defined by real-time monitoring of their distribution. In spite of repeated attempts to use Lipososmes for site-directed delivery of hydrophilic and hydrophobic drugs for cancer therapy, their application is limited due to poor understanding of lipososmes interactions with the cells and the sub-optimal biodistribution profile of various formulations. We will circumvent these problems using multifunctional liposomes with targeting, imaging and optimal drug release capabilities. Our strategy, involving assessment of target presence and distribution in an individual patient followed by optimized, target-specific drug delivery, may significantly improve efficacy of breast cancer treatment while reducing side effects. Experimental procedures Labeling with imaging agents The Affibody molecules are conjugated using maleimide chemistry with either AlexaFluor or 18F for, respectively, optical and PET imaging. Thermosensitive liposomes Liposomes with optimal lipid composition, sensitive to temperatures just above 37oC. The thermal destabilization of liposomes in a temperature range of 37oC-45oC will be determined by monitoring calcein leakage by spectrofluorometry methods. The next step will include optimization of techniques to conjugate HER2-specific Affibody molecules to the liposome surface via the maleimide group and their labeling with optical, PET, and MRI imaging agents. As the liposome size is an important determinant of their biodistribution, we will characterize the size of the resulting conjugates using the resources at Nanotechnology Characterization Laboratory. The liposomes will be loaded with therapeutic agents according to published methods. We will consult clinical oncologists at CCR regarding the best drugs available for treatment of breast cancers. In vitro and in vivo characterization Using binding, proliferation and clonogenic survival assays, as well as molecular biology methods we will thoroughly characterize in vitro the binding properties of the conjugates and their effects on the target cells. We will also investigate the impact of their binding on receptor expression and binding of therapeutic antibodies (Herceptin). Biodistribution of the conjugates will be studied using nude mice bearing xenografts of HER2-positive tumors. To test the in vivo imaging capacity of radioconjugates, we will monitor, by optical or PET imaging, the expected downregulation of HER2 in tumor xenografts following treatment with Herceptin or DMAG. The estimation of expression level obtained from imaging data will be verified by ex-vivo analysis of tumor tissue by immunohistochemistry and Western blots. Initial in vivo studies of our drug delivery system will be carried out using liposomes loaded with a combination of hydrophilic and hydrophobic fluorescent markers allowing to map and track in vivo tissue/organ compartmentalization of liposomes and to monitor drug leakage and/or release at 37oC-45oC. Noninvasive methods based on focused ultrasound techniques will be optimized to disintegrate liposomes. MRI, and micro-PET may be used to optimize the imaging capacity of the conjugates by those modalities. Experimental therapy of HER2-positive tumor-bearing animals will be carried out to assess the improvement of the efficacy of therapeutic agents delivered by tumor targeting, heat sensitive liposomes as compared with current application methods. For Exploratory IND Studies a single mammalian species can be used to establish a margin of safety. Project Status Probes for Optical Imaging Affibody-AlexaFluor conjugates have been developed and compared to Herceptin- AlexaFluor conjugates in vitro and in vivo. We showed in vitro that Affibody molecules bind to different epitomes of HER2 than does Herceptin and Affibody has only limited effect on HER2 signaling pathways as compared with Herceptin. Comparative in vivo imaging studies of AlexaFluor labeled ZHER2-Cys Affibody and Herceptin have been carried out using nude mice with human breast and ovarian cancer xenografts. While high tumor-specific accumulation of Heceptin was observed, only weak signal could be detected at the tumor, most likely, due to the fast clearance from the blood and high accumulation of AlexaFluor-labeled ZHER2-Cys Affibody in the kidneys. To increase the time of the Affibody molecules in the blood, molecules containing an albumin binding domain have been labeled with AlexaFluor. Biodistribution studies of these conjugates are carried out. Probes for PET Imaging Methods for 18F labeling of Affibody molecules are being developed. The results of in vitro studies showed that pre-incubation with non-labeled Affibody molecules inhibited the binding of the radioconjugates to the cells in a dose-dependent manner. Saturation analysis of His6-ZHer2:342-Cys-18F binding to the cell membranes indicated a single class of high-affinity binding sites. In vivo, high accumulation of the radioactivity in the tumor was observed as early as 20 min after injection as showed by PET imaging and did not reach the plateau after 60 min. That was confirmed by the results of the biodistribution studies indicating that, already 1 hr post-injection, the concentration of radioactivity (%ID/g) in tumor was approximately 10 times higher than that in the blood. Three hours later, that ratio increased to almost 17. On contrary, the initially high concentration in the kidneys (comparable to that in the tumor) decreased three fold at the same time. HER2-Specific Delivery System Conjugates of thermo-sensitive liposomes with HER2-specific, ZHER2-Cys Affibody molecules were characterized in vitro. We investigated the time-dependent uptake of Affibody-modified liposomes versus non modified liposomes in HER2-positive SKBR-3 cells and HER2-negative U251 cells. There was no significant difference liposome binding at two studied temperatures: 22oC and 37oC. After 30 min incubation, we were able to show 8-fold enrichment of Affibody-modified liposomes in HER-2-positive cells as comported to non-modified liposomes. With the decreasing concentration of Affibody we observed much lower non-specific banding. Cell fluorescence after 2h incubation with non-modified liposomes was comparable to untreated control [summary truncated at 7800 characters]