Molecular Imaging and Targeted Therapy of HER2-Positive Breast Cancers

背景和意义乳腺癌中HER 2受体的表达与不良预后相关，并且与原发肿瘤相比，它们在远处转移中的表达可能不同。该项目将提供评估乳腺癌（包括转移瘤）中HER 2总体表达的方法，并将治疗剂特异性地递送至HER 2阳性细胞。作为靶向剂，我们建议使用从我们在瑞典的CRADA合作伙伴（http：//www.example.com）获得的亲和体分子。www.affibody.com这些非常稳定且高度可溶的α-螺旋蛋白相对较小（8.3kDa），并且可以容易地在细菌系统中表达或通过肽合成产生。His 6-Zher 2：324以高亲和力（22 pM）结合HER 2受体，并且在羧基末端具有半胱氨酸以促进缀合。为了成像目的，这些分子用放射性核素或近红外光学信标标记。对于治疗，以根除转移性环境中的广泛疾病（其中即使具有已知位置的疾病也可能过于广泛而不能使用局部治疗方式）和用于在辅助环境中治疗以治疗不可检测的微转移的治疗，我们正在开发基于Affitoxins抗体的重组蛋白，其将Affibody的HER 2靶向能力与具有治疗潜力的蛋白质组合，例如，细菌毒素或促凋亡细胞信号分子。最初，专注于使用Affiprobes（含有荧光蛋白的分子）开发具有最佳亚细胞定位能力的蛋白质。然后，我们将测试适当的效应器，例如，PE 38和p53的分子定位在细胞质和细胞核中，分别。 我们的策略，包括评估目标的存在和分布在个别患者，然后优化，目标特异性药物输送，可以显着提高乳腺癌治疗的疗效，同时减少副作用。 使用成像剂标记亲和体分子使用马来酰亚胺化学与近红外荧光分子或正电子发射体缀合，分别用于光学和PET成像。 通过PCR扩增HA 22免疫毒素的PE 38部分，然后将PCR产物连接到含有在IPTG诱导型T7启动子控制下的HER 2-亲和体的载体中，开发并克隆了将HER 2-特异性亲和体分子与假单胞菌毒素组合的重组DNA构建体。类似的程序将用于产生含有不同效应物和亚细胞定位结构域的重组蛋白。体外和体内表征使用结合、增殖和克隆形成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。将使用携带HER 2阳性肿瘤异种移植物的裸鼠研究新型肿瘤靶向分子的生物分布。为了测试放射性缀合物的体内成像能力，我们通过光学和PET成像监测用赫赛汀或DMAG治疗后肿瘤异种移植物中HER 2的下调。从成像数据获得的表达水平的估计通过免疫组织化学和蛋白质印迹的肿瘤组织的离体分析来验证。使用共聚焦显微镜检测阿菲毒素的亚细胞分布，并在体外和体内评估其治疗功效。 成绩：1.我们已经证明AlexaFluor标记的亲和体分子可用于治疗干预后HER 2表达变化的体内定量。这项工作的结果已发表在Molecular Imaging上。2.我们已经将HER 2特异性亲和体分子与假单胞菌外毒素A（PE 38 KDEL）的截短和优化版本进行了基因融合。所得到的称为HER 2-Affitoxin的重组蛋白将Affibody分子的高HER 2特异性和亲和力与PE 38 KDEL的杀肿瘤潜力相结合。这项工作的结果发表在Journal of Immunotherapy上。3.我们已经创建了用于EGFR和HER 2体外染色的光稳定且相对简单生产的成像探针。这些新试剂称为Affiprobes，由靶向部分、HER 2或EGFR特异性Affibody分子和荧光部分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表明，高特异性和信号/背景比的Affiprobes。Affiprobes能够对活细胞和冷冻肿瘤干片进行染色。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。这项研究成果发表在European Journal of Chemical Biology：ChemBioChem上。4.已经开发了几种基于Affibody的含有荧光蛋白和不同亚细胞定位结构域的重组蛋白，并通过共聚焦显微镜研究了它们的运输。

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 are labeled with radionuclides or near-infrared optical beacons. For therapy, 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 are developing 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, focus on development of proteins with optimal of subcellular localization capacity using Affiprobes- molecules containing fluorescent proteins. Then we will test appropriate effectors, for instance, PE38 and p53 for molecules localizing in cytosol and nucleus, respectively. 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 near-infrared fluorescent molecules or positron emitters for, respectively, optical and PET imaging. Affitoxins A recombinant DNA construct combining HER2-specific Affibody molecules with Pseudomonas toxin hgave been 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 are monitoring, by optical and PET imaging, the downregulation of HER2 in tumor xenografts following treatment with Herceptin or DMAG. The estimation of expression level obtained from imaging data is verified by ex-vivo analysis of tumor tissue by immunohistochemistry and Western blots. The subcellular distribution of Affitoxin is tested using confocal microscopy and their therapeutic efficacy is assessed in vitro and in vivo. Accomplishments: 1. We have proved that AlexaFluor-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 Molecular Imaging. 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. The results of this work were published in 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. The results of this work were published in European Journal of Chemical Biology: ChemBioChem. 4. Several Affibody-based recombinant proteins containing fluorescent proteins and different sub-cellular localization domains have been developed and their trafficking studies by confocal microscopy.