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: 8349121
• 负责人: Jacek Capala
• 金额: $ 57.61万
• 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/8349121
• 关键词: Adjuvant; Adverse effects; Affect; Affinity; African American; Aggressive behavior; Alternative Therapies; American; Antigen Receptors; Apoptotic; Bacterial Toxins; Binding; Biodistribution; Biological Assay; Biology; Biophysics; Breast Cancer Treatment; Cell Nucleus; Cell Surface Receptors; Cells; Chemicals; Chemistry; Chemotherapy-Oncologic Procedure; Clinical; Confocal Microscopy; Cooperative Research and Development Agreement; Core Facility; Cytosol; Data; Development; Diagnosis; Discipline; Discipline of Nuclear Medicine; Disease; Distant Metastasis; Down-Regulation; Drug Delivery Systems; ERBB2 gene; Epidermal Growth Factor Receptor; European; Fever; Flow Cytometry; Freezing; Functional Imaging; Gene Expression; Gene Mutation; Growth; Growth Factor; Home environment; Image; Immunohistochemistry; Immunotoxins; In Vitro; Individual; Isopropyl Thiogalactoside; Joints; Journals; Label; Life; Ligation; Lipid Chemistry; Location; Lung; Magnetic Resonance Imaging; Maleimides; Malignant Neoplasms; Mammary Neoplasms; Manuscripts; Membrane; Methodology; Methods; Micrometastasis; Modality; Modification; Molecular Biology; Molecular Profiling; Molecular Target; Molecular and Cellular Biology; Monitor; Neoplasm Metastasis; Nude Mice; Optics; Outcome; Patients; Peptide Synthesis; Pharmaceutical Chemistry; Positron; Positron-Emission Tomography; Primary Neoplasm; Procedures; Production; Property; Protein Chemistry; Protein p53; Proteins; Pseudomonas; Pseudomonas aeruginosa toxA protein; Publishing; Radiation therapy; Radiation-Sensitizing Agents; Radiochemistry; Radioconjugate; Radioisotopes; Radiology Specialty; Reagent; Recombinant DNA; Recombinant Proteins; Refractory; Relapse; Research Design; Research Project Grants; Roche brand of trastuzumab; Signal Pathway; Signal Transduction; Signaling Molecule; Specificity; Staining method; Stains; Surface Antigens; Sweden; System; Systemic Therapy; TP53 gene; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Toxin; Tracer; Trastuzumab; Treatment Efficacy; Tumor Tissue; Tumor Volume; United States National Institutes of Health; Western Blotting; Widespread Disease; Woman; Work; Xenograft procedure; anticancer research; base; chemotherapy; expression cloning; high risk; image visualization; imaging probe; improved; in vivo; innovation; malignant breast neoplasm; molecular imaging; multimodality; news; novel; optical imaging; outcome forecast; overexpression; partial response; promoter; receptor; receptor expression; research study; response; targeted delivery; therapeutic target; therapy design; trafficking; triple-negative invasive breast carcinoma; tumor; tumor xenograft; vector

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.

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。 The other target would be EGFR. 尽管人们对 ER 阳性和 HER2 阳性乳腺癌生物学的理解取得了重大进展，导致针对此类患者的靶向治疗大幅增长，但针对 ER/PR/HER2 三阴性乳腺癌的进展却很少，此类乳腺癌占优先影响年轻和非裔美国女性的乳腺癌的 10-17%。这些肿瘤经常 (56-86%) 表达表皮生长因子受体 (EGFR)，并且与 HER2 过表达肿瘤一样，与所有乳腺癌亚型的侵袭行为和最差的预后相关，并且在前五年内会出现早期复发。尽管不是同义的，但大多数 TNBC 在基因表达阵列上携带类似基底细胞的分子谱，并且大多以高 p53 IHC 表达或 p53 基因突变为特征，这可能对化疗敏感性有影响。目前的数据表明，大多数 TNBC 患者对常规化疗方案是难治性的或仅表现出部分反应，并且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，并且尚无经过临床验证的靶向疗法。因此，针对这部分高危患者开发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，利用功能成像对 EGFR 进行可视化和定量，并结合 EGFR-Affibody 和修饰的 p53 肿瘤抑制因子进行治疗性重组蛋白进行靶向治疗，以改善 TNBC 的治疗效果。该项目将提供评估乳腺癌（包括转移瘤）中 HER2 或 EGFR 整体表达的方法，并向 HER2 和 EGFR 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 (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，这些分子被放射性核素或近红外光信标标记。对于治疗而言，为了根除转移环境中的广泛疾病（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），以及为了在辅助环境中治疗不可检测的微转移，我们正在开发基于 Affitoxins affibody 的重组蛋白，将 Affibody 的 HER2 靶向能力与具有治疗潜力的蛋白质（例如细菌毒素）相结合 or pro-apoptotic cell signaling molecules.最初，重点关注使用含有荧光蛋白的 Affiprobes 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子，例如分别定位于细胞质和细胞核中的分子的 PE38 和 p53。 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。 研究设计 用显像剂进行标记 Affibody 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器结合，分别用于光学和 PET 成像。 Affitoxins 通过 PCR 扩增 HA22 免疫毒素的 PE38 部分，然后将 PCR 产物连接到包含在 IPTG 诱导型 T7 启动子控制下的 HER2-affibody 的载体中，开发并克隆了将 HER2 特异性 Affibody 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子和亚细胞定位域的重组蛋白。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。将使用带有 HER2 阳性肿瘤异种移植物的裸鼠来研究新型肿瘤靶向分子的生物分布。为了测试放射性结合物的体内成像能力，我们通过光学和 PET 成像监测赫赛汀或 DMAG 治疗后肿瘤异种移植物中 HER2 的下调。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从成像数据获得的表达水平的估计。使用共聚焦显微镜测试 Affitoxin 的亚细胞分布，并在体外和体内评估其治疗功效。 成就： 1. 我们成功用正电子发射体 68-Ga 标记 HER2-Affibody，并在体外和体内验证了所得示踪剂。研究结果发表在《欧洲核医学和分子成像杂志》上。 2. 我们已经证明，AlexaFluor 标记的 Affibody 分子可用于体内量化治疗干预后 HER2 表达变化。这项工作的结果发表在《分子影像》杂志上。 3. 我们将 HER2 特异性 Affibody 分子与假单胞菌外毒素 A (PE38KDEL) 的截短和优化版本进行基因融合。由此产生的重组蛋白称为 HER2-Affitoxin，将 Affibody 分子的高 HER2 特异性和亲和力与 PE38KDEL 的杀肿瘤潜力结合在一起。这项工作的结果发表在《临床癌症研究》上，并在美国癌症研究协会最近的新闻稿中进行了专题介绍http://www.aacr.org/home/public--media/aacr-in-the-news.aspx?d=2421。 4. 我们已经创建了光稳定且相对简单生产的成像探针，用于 EGFR 和 HER2 的体外染色。这些新试剂称为 Affiprobes，由靶向部分、HER2 或 EGFR 特异性 Affibody 分子以及荧光部分 mCherry（红色）或 EGFP（绿色）组成。我们的流式细胞术和共焦显微镜实验证明了 Affiprobes 的高特异性和信号/背景比。 Affiprobes 能够对活细胞和冷冻肿瘤静电切片进行染色。这种类型的光学探针可以轻松扩展以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. 已经开发了几种含有荧光蛋白和不同亚细胞定位结构域的基于 Affibody 的重组蛋白，并通过共聚焦显微镜对其运输进行了研究。 6. 我们已成功将18-F标记的Affibody应用于结合PET、CT和MRI的多模态成像环境中，以检测动物肺部的微转移。由此产生的手稿已提交给临床癌症研究。 7. 我们已成功应用 18-F 标记的 Affibody 来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的早期肿瘤反应。由此产生的手稿已提交给临床癌症研究。

项目成果

Changes in HER2 expression in breast cancer xenografts after therapy can be quantified using PET and (18)F-labeled affibody molecules.

• DOI: 10.2967/jnumed.108.057695
• 发表时间: 2009-07
• 期刊: Journal of nuclear medicine : official publication, Society of Nuclear Medicine
• 作者: Kramer-Marek G;Kiesewetter DO;Capala J
• 通讯作者: Capala J

The crosstalk between EGF, IGF, and Insulin cell signaling pathways--computational and experimental analysis.

• DOI: 10.1186/1752-0509-3-88
• 发表时间: 2009-09-04
• 期刊: BMC systems biology
• 作者: Zielinski R;Przytycki PF;Zheng J;Zhang D;Przytycka TM;Capala J
• 通讯作者: Capala J

Quantitative Analysis of HER2 Receptor Expression In Vivo by Near-Infrared Optical Imaging

• DOI: 10.2310/7290.2010.00018
• 发表时间: 2010-07-01
• 期刊: MOLECULAR IMAGING
• 影响因子: 2.8
• 作者: Chernomordik, Victor;Hassan, Moinuddin;Capala, Jacek
• 通讯作者: Capala, Jacek

HER2-affitoxin: a potent therapeutic agent for the treatment of HER2-overexpressing tumors.

• DOI: 10.1158/1078-0432.ccr-10-2887
• 发表时间: 2011-08-01
• 期刊: Clinical cancer research : an official journal of the American Association for Cancer Research
• 作者: Zielinski R;Lyakhov I;Hassan M;Kuban M;Shafer-Weaver K;Gandjbakhche A;Capala J
• 通讯作者: Capala J

Sarcosine induces increase in HER2/neu expression in androgen-dependent prostate cancer cells.

• DOI: 10.1007/s11033-010-0442-2
• 发表时间: 2011-10
• 期刊: Molecular biology reports
• 影响因子: 2.8
• 作者: Dahl M;Bouchelouche P;Kramer-Marek G;Capala J;Nordling J;Bouchelouche K
• 通讯作者: Bouchelouche K