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）。这些非常稳定且高度可溶&amp；#945；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。治疗将采用两种不同的方法。1. 为了靶向可见的、明确的肿瘤，his6 - zhen2:324将与热敏脂质体偶联，这些脂质体被标记为用于体内成像的beacon，并装载治疗药物（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。2. 为了根除转移性环境中的广泛疾病（即使是已知位置的疾病也可能过于广泛而无法使用局部治疗方式）以及在辅助治疗环境中治疗无法检测到的微转移，我们将开发基于Affitoxins的重组蛋白，将附着体的her2靶向能力与具有治疗潜力的蛋白质（例如细菌毒素或促凋亡细胞信号分子）结合起来。首先，我们将重点开发具有最佳亚细胞定位能力的蛋白质，使用含有荧光蛋白的亲和分子。然后，我们将测试适当的效应物，例如，PE38和p53分别定位于细胞质和细胞核中的分子。我与勒纳尔研究所的Peter Chumakov博士的合作将促进后期治疗模式的研究，他将与我分享他实验室开发的试剂和实验工具，包括各种表达结构的杂交p53 cDNA和p53功能活性的高通量测试系统（基于慢病毒的报告系统，可以方便地测量几乎任何细胞类型中p53的转录活性）。包括各种体内模型)。这种方法将有助于早期识别最有希望的蛋白质，从而将精力集中在最有希望的化合物上。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。用显像剂标记研究设计附体分子使用马来酰亚胺化学与AlexaFluor或18F偶联，分别用于光学和PET成像。这项任务将与CCR纳米生物学项目的Robert Blumenthal博士和他的团队合作进行。脂质体具有最佳的脂质组成，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。在iptg诱导的T7启动子控制下，通过PCR扩增HA22免疫毒素PE38部分，将PCR产物连接到含有her2附着体的载体上，构建her2特异性附着体分子与假单胞菌毒素结合的重组DNA构建体。类似的程序将用于生产含有不同效应器和亚细胞定位域的重组蛋白。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。新的肿瘤靶向分子的生物分布将在携带her2阳性肿瘤异种移植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。将使用共聚焦显微镜检测Affitoxin的亚细胞分布，并在体外和体内评估其治疗效果。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。我们已经证明，f -18标记的附着体分子可以用于体内定量治疗干预后HER2表达的变化。这项工作的结果已经发表在《核医学杂志》上，该杂志的一篇社论以及美国国立卫生研究院和美国核学会的新闻稿都强调了它们的重要性。我们将her2特异性的附着体分子与假单胞菌外毒素a （PE38KDEL）的截断和优化版本进行了基因融合。由此产生的重组蛋白HER2- affitoxin结合了高HER2特异性和附着体分子的亲和力以及PE38KDEL的肿瘤杀伤潜力。报道这种新型重组抗癌药物的克隆、过表达、纯化和体外鉴定的手稿已被《免疫治疗杂志》接受发表。我们已经创建了光稳定和相对简单的成像探针，用于体外染色EGFR和HER2。这些新试剂被称为词缀探针，由靶向片段、HER2或egfr特异性词缀分子和荧光片段mCherry（红色）或EGFP（绿色）组成。我们的流式细胞术和共聚焦显微镜实验证明了Affiprobes的高特异性和信号/背景比。粘附剂既能染色活细胞，也能染色冷冻肿瘤切片。这种类型的光学探针可以很容易地扩展到靶向其他细胞表面抗原/受体。描述这项工作的手稿已提交给欧洲化学生物学杂志：ChemBioChem。4. 几种含有荧光p的基于抗体的重组蛋白[摘要截短于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. Two different approaches will be used for therapy. 1. To target visible, well defined tumors, 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. 2. To eradicate widespread disease in the metastatic setting (in which even the disease with known locations may be too widespread to use local treatment modalities) and for treatment in the adjuvant setting to treat non-detectable micrometastases we will develop Affitoxins affibody-based recombinant proteins combining HER2-targeting capacity of Affibody with proteins having therapeutic potential such as, for example, bacterial toxins or pro-apoptotic cell signaling molecules. Initially well focus on development of proteins with optimal of subcellular localization capacity using Affiprobes- molecules contain fluorescent proteins. Then we will test appropriate effectors, for instance, PE38 and p53 for molecules localizing in cytosol and nucleus, respectively. Investigation of the later therapeutic modality will be facilitated by my collaboration with Dr. Peter Chumakov of the Learner Research Institute, who will share with me reagents and experimental tools developed in his laboratory including the hybrid p53 cDNA in various expression constructs and a high throughput test system for functional activity of p53 (lentivirus-based reporter systems that conveniently measure transcriptional activity of p53 in virtually any cell type, including various in vivo models). This approach will facilitate early identification of the most promising protein allowing focusing the effort on the most promising compounds. 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. Research Design 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 This task will be carried oput in collaboration with Dr. Robert Blumenthal and his team at CCR Nanobiology Program. 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. Affitoxins A recombinant DNA construct combining HER2-specific Affibody molecules with Pseudomonas toxin will be developed and cloned by PCR amplification of the PE38 part of HA22 immunotoxin following by ligation of the PCR product into vector containing the HER2-affibody under control of IPTG-inducible T7 promoter. Similar procedures will be used to produce recombinant proteins containing different effectors and sub-cellular localization domains. 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. Biodistribution of the novel tumor-targeted molecules 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. 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. The subcellular distribution of Affitoxin will be tested using confocal microscopy and their therapeutic efficacy will be assessed in vitro and in vivo. For Exploratory IND Studies a single mammalian species can be used to establish a margin of safety. Accomplishments: 1. We have proved that F-18-labeled Affibody molecules can be used for in vivo quantification of changes in HER2 expression following therapeutic intervention. The results of this work have been published in the Journal of Nuclear Medicine and their significance highlighted by an editorial in the same journal as well as press releases from NIH and American Nuclear Society. 2. We have genetically fused HER2-specific Affibody molecule with a truncated and optimized version of Pseudomonas Exotoxin A (PE38KDEL. The resulting recombinant protein called HER2-Affitoxin combines high HER2 specificity and affinity of Affibody molecules with the tumoricidal potential of PE38KDEL. Manuscript reporting cloning, overexpression, purification, and in vitro characterization of this novel recombinant anti-cancer agent has been accepted for publication in the Journal of Immunotherapy. 3. We have created photo-stable and relatively simple-to-produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called Affiprobes, consist of a targeting moiety, a HER2- or EGFR-specific Affibody molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of Affiprobes. Affiprobes are able to stain both live cells and frozen tumor xerograph sections. This type of optical probe can easily be extended for targeting other cell-surface antigens/receptors. A manuscript describing this work has been submitted to a European Journal of Chemical Biology: ChemBioChem. 4. Several Affibody-based recombinant proteins containing fluorescent p [summary truncated at 7800 characters]