Chemically Controlled Assembly of Therapuetic Protein Nanostrctures

描述（由申请人提供）：设计和开发既能作为生物传感器又能作为治疗剂的纳米器件，将极大地推进包括癌症在内的许多人类疾病的治疗。由于单克隆和重组抗体已经被用于向癌症组织输送各种毒素、药物和放射性核素，因此它们可以被用作抗癌纳米结构的识别配体。放射性核素通过偶联抗体靶向肿瘤已被证明是一种成功的成像和抗肿瘤方法。尽管单克隆抗体放射性核素偶联物在临床前和临床都取得了成功，但其发展仍面临许多问题，如肾清除率低导致成像能力差、放射性核素骨沉积有毒、偶联化学与免疫反应性不相容以及分子异质性。我们建议开发一种具有双特异性的多价放射免疫治疗纳米结构的药理学控制组装和拆卸方案。我们将利用我们最近发现的如何从大肠杆菌二氢叶酸还原酶（DHFR2）融合蛋白中构建离散的化学诱导蛋白纳米结构（8-30 nm直径）。我们将制备DHFR2分子与Daniel Vallera博士（合作研究者）开发的单链抗体（scFv）融合，并结合b细胞淋巴瘤和白血病抗原CD22。我们将制备dhfr2 -抗cd22 scFv的融合蛋白，这些融合蛋白能够在甲氨蝶呤二聚体与荧光基团或螯合放射性核素偶联的情况下自组装成二价、四价或八价物种。我们将证明抗体纳米蛋白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粒在体内的生物分布，以及timethoprim（一种无毒的大肠杆菌DHFR抑制剂）在体内促进低聚物分解的能力。我们还将在小鼠异种移植肿瘤模型上研究dhfr2 -抗cd22 scFv纳米颗粒的抗肿瘤和肿瘤成像特性。利用分子建模和蛋白质工程原理，我们将设计能够异源二聚化的DHFR分子。这些突变DHFRs将在未来的研究中用于制备自组装双特异性抗体纳米粒。本研究阐明的原理将适用于未来能够检测和治疗多种癌症的抗体-放射性核素纳米结构的设计。我们研究的目标是开发纳米粒子，使其能够锁定肿瘤，报告肿瘤的位置并摧毁肿瘤。在我们的第一次尝试中，我们将开发一种方法来制备可靶向B细胞白血病的放射性标记抗体蛋白纳米粒。我们将把这些抗体纳米颗粒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.