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。另一个目标是EGFR。虽然在了解ER阳性和HER2阳性乳腺癌的生物学方面已经取得了重大进展，这导致了针对这组患者的靶向治疗的巨大增长，但对于ER/PR/HER2三阴性乳腺癌却很少有这样的进展，这代表了10-17%的乳腺癌，优先影响年轻和非裔美国女性。这些肿瘤经常（56-86%）表达表皮生长因子受体（EGFR），并且与HER2过表达的肿瘤一样，在所有乳腺癌亚型中具有侵袭性行为和最差的预后，并且在前5年内早期复发。虽然不是同义词，但大多数TNBC在基因表达阵列上携带基底样分子谱，并且大多数以p53 IHC高表达或p53基因突变为特征，这可能与化疗敏感性有关。目前的数据表明，大多数TNBC患者将是难治性的或对传统化疗方案仅表现出部分反应，并将在诊断后的最初几年内死于转移性疾病。值得注意的是，与其他亚型不同，化疗是目前三阴性乳腺肿瘤患者唯一的全身治疗方式，目前尚无临床验证的靶向治疗方法。因此，开发针对这部分高危患者的新型靶向疗法至关重要。因此，我们提出了一个双管齐下的研究项目，将利用功能成像可视化和定量的EGFR和治疗重组蛋白结合EGFR-附着体和修饰p53肿瘤抑制因子靶向治疗，以改善TNBC治疗的结果。该项目将为评估乳腺癌（包括转移）中HER2或EGFR的全球表达提供手段，并为HER2和EGFR阳性细胞提供专门的治疗药物。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，并在羧基末端与半胱氨酸结合以促进结合。为了成像的目的，这些分子被放射性核素或近红外光学信标标记。在治疗方面，为了根除转移性疾病中的广泛传播（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），以及在辅助治疗方面治疗无法检测到的微转移，我们正在开发基于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。最初，重点是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使用马来酰亚胺化学将附着体分子与近红外荧光分子或正电子发射器偶联，分别用于光学和PET成像。在iptg诱导的T7启动子控制下，通过PCR扩增HA22免疫毒素的PE38部分，并将PCR产物连接到含有her2粘附体的载体上，构建了一个将her2特异性粘附体分子与假单胞菌毒素hgave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，我们通过光学和PET成像监测HER2在接受赫赛汀或DMAG治疗后的肿瘤异种移植物中的下调。通过免疫组织化学和Western blot对肿瘤组织进行离体分析，验证了从影像学数据中获得的表达水平的估计。用共聚焦显微镜检测了黄曲霉毒素的亚细胞分布，并对其体内和体外的治疗效果进行了评价。成就:1。我们成功地用正电子发射器68-Ga标记了her2 -附着体，并在体外和体内验证了所得到的示踪剂。研究结果发表在《欧洲核医学和分子成像杂志》上。我们已经证明，alexafluor标记的固定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Molecular Imaging杂志上。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。这项工作的结果发表在《临床癌症研究》杂志上，并在美国癌症研究协会最近的一份新闻稿中有所介绍。4. 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。这项工作的结果发表在欧洲化学生物学杂志：ChemBioChem上。5. 几种含有荧光蛋白和不同亚细胞定位结构域的重组蛋白已被开发出来，并在共聚焦显微镜下进行了转运研究。6. 我们成功地将18f - labeled Affibody应用于PET， CT和MRI的多模态成像设置中，以检测肺部的微转移。最终的手稿已提交给临床癌症研究。我们已成功应用18-F-Labeled affenbody研究曲妥珠单抗治疗期间her2表达的体内变化，并表明这些变化可用于预测曲妥珠单抗对早期肿瘤的反应。最终的手稿已提交给临床癌症研究。

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. The other target would be EGFR. Although a significant progress has been made in the understanding of the biology of ER-positive and HER2-positive breast cancers, which has led to the enormous growth in targeted therapeutic for this group of patients, few such developments have been made for ER/PR/HER2 triple negative breast cancer, which represent 10-17% of breast cancers that preferentially affect young and African-American women. The tumors frequently (56-86%) express epidermal growth factor receptor (EGFR) and, like HER2 overexpressing tumors, are associated with aggressive behavior and the poorest prognosis of all breast cancer subtypes with early relapses within the first five years. Although not synonymous, the majorities of TNBC carry the basal-like molecular profile on gene expression arrays and mostly are characterized by high p53 IHC expression or p53 gene mutations, which may have implications for chemotherapy sensitivity. Current data suggest that the majority of TNBC patients will be refractory or show only a partial response to conventional chemotherapy regimens and will die of metastatic disease within the first few years after diagnosis. It is noteworthy that, unlike for other subtypes, chemotherapy is currently the only modality of systemic therapy for patients bearing triple negative breast tumors and there is no clinically validated targeted therapy for their treatment. Therefore, the development of novel targeted therapies for this subset of high-risk patients is of paramount importance. Therefore, we propose a two-pronged research project that will utilize functional imaging for visualization and quantification of EGFR and therapeutic recombinant protein combining EGFR-Affibody and modified p53 tumor suppressor for targeted therapy to improve outcome of TNBC treatment. This project will provide means to assess global expression of HER2 or EGFR in breast cancers (including metastases) and to deliver therapeutic agents specifically to HER2 and EGFR-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 successfully labeled HER2-Affibody with a positron emitter, 68-Ga and validated the resulting tracer in vitro and in vivo. The results were published in European Journal of Nuclear Medicine and Molecular Imaging. 2. 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. 3. 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 Clinical Cancer Research and were featured in a recent pres release from American Association for Cancer Researchhttp://www.aacr.org/home/public--media/aacr-in-the-news.aspx?d=2421. 4. 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. 5. Several Affibody-based recombinant proteins containing fluorescent proteins and different sub-cellular localization domains have been developed and their trafficking studies by confocal microscopy. 6. We have successfully applied 18-F-Labeled Affibody in multimodality imaging setting combining PET, CT and MRI, to detect beast micrometastases in the lungs. The resulting manuscript has been submitted to Clinical Cancer Research. 7. We have successfully applied 18-F-Labeled Affibody to study in vivo changes of HER2-expression during treatment with trastuzumab and showed that those changes might be used to predict early tumor response to trastuzumab. The resulting manuscript has been submitted to Clinical Cancer Research.