Chemically Controlled Assembly of Therapuetic Protein Nanostrctures

描述（由申请人提供）：能够同时用作生物传感器和治疗剂的纳米器件的设计和开发将极大地推进许多人类疾病（包括癌症）的治疗。因为它们已经被用于将各种毒素、药物和放射性核素递送到癌症组织，所以单克隆抗体和重组抗体可以被利用作为抗癌纳米结构的识别配体。通过与抗体缀合将放射性核素靶向肿瘤已被证明是一种成功的成像和抗肿瘤方法。尽管它们在临床前和临床上取得了成功，但单克隆抗体放射性核素缀合物的开发受到许多问题的困扰，例如，由于肾清除率低而导致的成像能力差、毒性放射性核素骨沉积、与免疫反应性和分子异质性不相容的缀合化学。我们建议开发一个协议的多价放射免疫纳米结构，具有潜在的双特异性的放射免疫控制的组装和拆卸。我们将利用我们最近的发现，如何构建离散的化学诱导蛋白质纳米环（8-30纳米直径）。来自大肠大肠杆菌二氢叶酸还原酶（DHFR 2）融合蛋白。我们将制备与单链抗体（scFv）融合的DHFR 2分子，该单链抗体由丹尼尔瓦莱拉博士（共同研究者）开发，并与B细胞淋巴瘤和白血病抗原CD 22结合。我们将制备DHFR 2-抗-CD 22 scFv的融合蛋白，其能够在与荧光团或螯合的放射性核素偶联的甲氨蝶呤二聚体存在下自组装成二价、四价或八价物质。我们将证明抗体纳米环能够选择性地结合并在体外进行细胞内B-白血病细胞的摄取和运输。此外，我们将确定抗体纳米环的体内生物分布，以及甲氧苄氨嘧啶，一种无毒的E。coli DHFR抑制剂，促进体内寡聚体分解。我们还将用小鼠异种移植肿瘤模型研究DHFR 2-抗CD 22 scFv纳米环的抗肿瘤和肿瘤成像特性。利用分子模拟和蛋白质工程原理，我们将设计能够异源二聚化的DHFR分子。这些突变的DHFR将用于未来的研究，以制备自组装双特异性抗体纳米环。本研究所阐明的原理将适用于设计抗体-放射性核素纳米环，这些纳米环能够在未来检测和治疗多种癌症。开发能够定位肿瘤、报告肿瘤位置并摧毁肿瘤的纳米颗粒是我们的研究目标。在我们的第一次尝试中，我们将开发一种方法来制备放射性标记的抗体蛋白纳米环，可以靶向B细胞白血病。我们将使用这些抗体纳米环用于肿瘤成像和抗肿瘤治疗，并证明我们可以在需要时去除纳米颗粒。

DESCRIPTION (provided by applicant): The design and development of nanodevices capable of functioning both as biosensors and therapeutic agents would greatly advance the treatment of a number of human diseases, including cancer. Because they have already been used to deliver a variety of toxins, drugs and radionuclides to cancer tissues, monoclonal and recombinant antibodies could be harnessed as recognition ligands for anticancer nanostructures. The targeting of radionuclides to tumors by conjugation to antibodies has been shown to be a successful imaging and antitumor approach. Despite their preclinical and clinical success, the development of monoclonal antibody radionuclide conjugates suffers from a number of concerns, such as, poor imaging capability due to low renal clearance, toxic radionuclide bone deposition, conjugation chemistry that is incompatible with immunoreactivity and molecular heterogeneity. We propose to develop a protocol for the pharmacologically controlled assembly and disassembly of multivalent radioimmunotherapeutic nanostructures that have the potential for bispecificity. We will take advantage of our recent discovery of how to construct discrete chemically induced protein nanorings (8-30 nm dia.) from E. coli dihydrofolate reductase (DHFR2) fusion proteins. We will prepare DHFR2 molecules fused to a single chain antibody (scFv) that was developed by Dr. Daniel Vallera (co-Investigator) and binds to the B-cell lymphoma and leukemia antigen CD22. We will prepare DHFR2-anti-CD22 scFv's fusion proteins that are able to self-assemble into bivalent, tetravalent or octavalent species in the presence of a methotrexate dimerizer coupled to a fluorophore or chelated radionuclides. We will demonstrate that the antibody-nanorings are able to selectively bind and undergo intracellular B-leukemia cell uptake and trafficking in vitro. In addition, we will determine the in vivo biodistribution of the antibody nanorings, as well as the ability of timethoprim, a non-toxic E. coli DHFR inhibitor, to promote oligomer disassembly in vivo. We will also investigate the anti-tumor and tumor imaging properties of the DHFR2-anti-CD22 scFv nanorings with a mouse xenograft tumor model. Using molecular modeling and protein engineering principles, we will design DHFR molecules capable of heterodimerization. These mutant DHFRs will be used in future studies to prepare self-assembling bispecific antibody nanorings. The principles elucidated by this study will be applicable to the design of antibody-radionuclide nanorings that are capable of detecting and treating a wide range of cancers in the future.The development of nanoparticles that can home in on a tumor, report back on where the tumor is and destroy the tumor is the goal of our research. In our first attempt, we will develop a method to prepare radiolabeled antibody protein nanorings that can target B- cell leukemias. We will use these antibody-nanorings for both tumor imaging and antitumor therapy and demonstrate that we can remove the nanoparticles when needed.