Molecular Imaging and Targeted Therapy of HER2-Positive Breast Cancers

HER2 阳性乳腺癌的分子影像和靶向治疗

• 批准号: 7733174
• 负责人: Jacek Capala
• 金额: $ 65.42万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733174
• 关键词: 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin; Adverse effects; Affinity; Albumins; Alternative Therapies; Animals; Antibodies; Binding; Biodistribution; Biological Assay; Biophysics; Breast Cancer Treatment; CCR; Cell Surface Receptors; Cells; Characteristics; Chemicals; Chemistry; Clinical; Complement; Consult; Cooperative Research and Development Agreement; Data; Dependence; Development; Discipline; Discipline of Nuclear Medicine; Distant Metastasis; Down-Regulation; Drug Delivery Systems; Drug Formulations; Drug Monitoring; Dyes; EGF Signaling Pathway; ERBB2 gene; Epitopes; European; Extravasation; Fluorescence Spectrometry; Fluorine; Focused Ultrasound Therapy; Growth Factor; Heating; Hydrophobicity; Image; Immunoglobulin Fragments; Immunohistochemistry; Immunology; In Vitro; Individual; Invasive; Investigational Therapies; Joints; Journals; Label; Laboratories; Lipid Chemistry; Lipids; Liposomes; Magnetic Resonance Imaging; Maleimides; Manuscripts; Maps; Membrane; Methodology; Methods; Modality; Modeling; Molecular Biology; Monitor; Mus; Nanotechnology; Neoplasm Metastasis; Nude Mice; Oncologist; Optics; Organ; Patients; Peptide Synthesis; Pharmaceutical Chemistry; Pharmaceutical Preparations; Positron-Emission Tomography; Primary Neoplasm; Primates; Procedures; Property; Protein Chemistry; Proteins; Publications; Publishing; Purpose; Radiation Oncology; Radiation-Sensitizing Agents; Radiochemistry; Radioconjugate; Radioisotopes; Radiology Specialty; Range; Receptor Down-Regulation; Reporting; Research; Resources; Roche brand of trastuzumab; Safety; Signal Pathway; Signal Transduction; Site; Surface; Sweden; System; Techniques; Temperature; Testing; Therapeutic; Therapeutic Agents; Therapeutic antibodies; Time; Tissues; Toxin; Tumor Tissue; Western Blotting; Xenograft procedure; anticancer research; base; cancer cell; cancer therapy; design; dimer; dosage; fluorexon; improved; in vivo; innovation; kinase inhibitor; malignant breast neoplasm; molecular imaging; monomer; nanobiology; nanoparticle; outcome forecast; programs; receptor; receptor expression; response; size; subcutaneous; technique development; therapy design; tumor; tumor xenograft

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 alpha-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. Accomplishments 1. Methods for labeling of HER2-specific Affibody molecules with fluorine-18 have been developed and published in the Journal of Fluorine Chemistry. 2. 18F-Affibody conjugates have been characterized in vitro and in vivo as reported in the European Journal of Nuclear Medicine and Molecular Imaging. 3. Different types of HER2-specific Affibody molecules (monomer, dimer, and albumin binding domain containing) have been labeled with AlexaFluor dyes and characterized in vitro and in vivo as reported in Clinical Cancer Research. 4. Thermosensitive liposomes have been developed and conjugated with HER2-specific Affibody molecules. A manuscript describing the design and physico-chemical characteristics of the resulting nanoparticles has been accepted for publication in the Journal of Liposome Research 5. Biodistribution studies of 18F-labeled Affibody in primates have been performed. 6. Subcutaneous models of tumors with different levels of HER2 expression have been established and used to assess the dependence of signal observed in imaging studies on the HER2 expression. 7. PET imaging was successfully used to quantify changes in HER2 expression following treatment of tumor bearing mice with 17-DMAG and the resulting manuscripot have been submitted for publication in the Journal of Nuclear Medicine.

背景和意义 乳腺癌中 HER2 受体的表达与不良预后相关，并且与原发肿瘤相比，其在远处转移灶中的表达可能有所不同。该项目将提供评估乳腺癌（包括转移瘤）中 HER2 整体表达的方法，并专门向 HER2 阳性细胞输送治疗药物。作为靶向剂，我们建议使用从瑞典 CRADA 合作伙伴 (http://www.affibody.com) 获得的 Affibody 分子。这些非常稳定且高度可溶的 α 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，并可在羧基末端与半胱氨酸结合以促进缀合。为了成像目的，这些分子用放射性核素标记。对于治疗，His6-Zher2:324 将与热敏脂质体结合，用信标标记用于体内成像并装载治疗剂（例如毒素、放射增敏剂或激酶抑制剂），通过实时监测药物分布来实现局部药物释放。尽管多次尝试使用脂质体定点递送亲水性和疏水性药物来治疗癌症，但由于对脂质体与细胞相互作用的了解不足以及各种制剂的次优生物分布特征，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个体患者中靶点的存在和分布，然后进行优化的靶点特异性药物输送，可以显着提高乳腺癌治疗的疗效，同时减少副作用。 实验程序 用显像剂标记 Affibody 分子使用马来酰亚胺化学与 AlexaFluor 或 18F 结合，分别用于光学和 PET 成像。热敏脂质体 具有最佳脂质成分的脂质体，对 37oC 以上的温度敏感。脂质体在 37°C-45°C 温度范围内的热失稳将通过分光荧光法监测钙黄绿素泄漏来确定。 下一步将包括优化技术，通过马来酰亚胺基团将 HER2 特异性 Affibody 分子缀合到脂质体表面，并用光学、PET 和 MRI 成像剂进行标记。由于脂质体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来表征所得缀合物的大小。脂质体将根据已发表的方法装载治疗剂。我们将咨询 CCR 的临床肿瘤学家，了解治疗乳腺癌的最佳药物。体外和体内表征利用结合、增殖和克隆存活测定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。我们还将研究它们的结合对受体表达和治疗抗体（赫赛汀）结合的影响。 将使用带有 HER2 阳性肿瘤异种移植物的裸鼠来研究缀合物的生物分布。为了测试放射性结合物的体内成像能力，我们将通过光学或 PET 成像监测赫赛汀或 DMAG 治疗后肿瘤异种移植物中 HER2 的预期下调。从成像数据获得的表达水平的估计将通过免疫组织化学和蛋白质印迹对肿瘤组织进行离体分析来验证。我们的药物递送系统的初步体内研究将使用装载有亲水性和疏水性荧光标记组合的脂质体进行，从而可以绘制和跟踪脂质体的体内组织/器官区室化，并在 37oC-45oC 下监测药物泄漏和/或释放。基于聚焦超声技术的非侵入性方法将被优化以分解脂质体。 MRI和微型PET可用于通过这些方式优化缀合物的成像能力。将进行HER2阳性荷瘤动物的实验治疗，以评估与现有应用方法相比，通过肿瘤靶向热敏脂质体递送的治疗剂的疗效的改善。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。成就 1. 已开发出用 18 氟标记 HER2 特异性 Affibody 分子的方法，并发表在《Journal of Fluorine Chemistry》上。 2. 18F-Affibody 缀合物已在体外和体内进行了表征，如《欧洲核医学和分子成像杂志》中报道的那样。 3. 不同类型的 HER2 特异性 Affibody 分子（单体、二聚体和含有白蛋白结合域）已用 AlexaFluor 染料进行标记，并按照临床癌症研究中的报告进行了体外和体内表征。 4. 已开发出热敏脂质体，并与 HER2 特异性 Affibody 分子结合。描述所得纳米颗粒的设计和物理化学特性的手稿已被《脂质体研究杂志》5 接受发表。18F 标记的 Affibody 在灵长类动物中的生物分布研究已经进行。 6.已经建立了具有不同HER2表达水平的肿瘤的皮下模型，并用于评估成像研究中观察到的信号对HER2表达的依赖性。 7. PET 成像已成功用于量化用 17-DMAG 治疗荷瘤小鼠后 HER2 表达的变化，所得手稿已提交在《核医学杂志》上发表。

项目成果

In vivo method to monitor changes in HER2 expression using near-infrared fluorescence imaging.

使用近红外荧光成像监测 HER2 表达变化的体内方法。

• 发表时间: 2012
• 期刊: Molecular imaging
• 影响因子: 2.8
• 作者: Hassan,Moinuddin;Chernomordik,Victor;Zielinski,Rafal;Ardeshirpour,Yasaman;Capala,Jacek;Gandjbakhche,Amir
• 通讯作者: Gandjbakhche,Amir