Tunable affinity and heat stable antibody targeting of nanoparticles

描述(申请人提供)：我们建议的研究将证明，我们可以通过可变淋巴细胞受体(VLR)抗体的纳米工程来生产一种能够同时具有可调亲和力和热稳定性的靶向载体。我们将通过VLR功能化纳米/微米颗粒的结合分析来展示这些靶向特性。我们将创造多价VLR抗体，具有不同的价态、接头长度，并具有适合附着在纳米/微米颗粒上的残基。VLRS的多价重组形式将识别称为BCLA的炭疽芽孢杆菌孢子壳蛋白。我们将通过生物物理技术和单分子成像来验证正确的价态和连接子排列。我们将使用表面等离子体共振(SPR)和基于原子力显微镜的单分子结合测量来测量VLR抗体与BCLA抗原的结合强度。我们将测量多价抗体的动力学速率和由此产生的亲和力，作为价态和连接物长度的函数。我们还将验证即使在高温下也会发生抗原结合。我们将利用高通量流式细胞术将多价VLRs偶联到几个微米/纳米颗粒上，并验证其与炭疽杆菌的粘附性。我们将测量作为a)VLR分子价态、b)VLR连接基间距、c)颗粒大小和d)温度的颗粒粘附性和凝聚性。我们的目标是通过1)确定设计原则，通过简单的价态改变提供一种合理的方法来控制分子亲和力，以及2)利用热稳定到70℃或更高温度的抗体分子，来扩大纳米颗粒针对带有抗体的病原体的用途。这项提议的成功完成将开辟几条卓有成效的发现之路，从而改进病原体和生物杀菌剂的传感器和对策。我们预计会产生具有可调节的抗原亲和力和热稳定性的纳米/微米颗粒。这两个影响将极大地提高基于抗体的检测器、对策和诊断方法的生存能力。 与公共卫生相关：我们建议创造新发现的VLR抗体靶向的纳米/微米颗粒。我们将创造重组形式的VLRs，我们预计将为药物输送、试剂开发和传感器提供以下好处，包括合理调整抗体亲和力和温度稳定性。

DESCRIPTION (provided by applicant): Our proposed study will demonstrate that we can produce a targeting vehicle capable of both tunable affinity and heat-stability through nanoscale engineering of Variable Lymphocyte Receptors (VLR) antibodies. We will demonstrate these targeting qualities through binding assays of VLR functionalized nano/microparticles. We will create multivalent VLR antibodies with a range of valencies, linker lengths, and that have suitable residues for attachment to nano/microparticles. The multivalent recombinant forms of VLRs will recognize the Bacillus anthracis spore coat protein called BclA. We will verify the proper valency and linker arrangement with biophysical techniques and single molecule imaging. We will measure the binding strength the VLR antibodies to the BclA antigen with surface plasmon resonance (SPR) and single-molecule binding measurements based upon atomic force microscopy force spectroscopy. We will measure the kinetic rates and the resulting affinity of the multivalent antibodies as a function of valency and linker length. We will also validate that antigen binding occurs even when raised to high temperatures. We will conjugate the multivalent VLRs to several micro/nanoparticles and validate adhesion to B. anthracis using high throughput flow cytometry. We will measure particle adhesion and agglutination as a function of a) VLR molecular valency, b) VLR linker spacing, c) particle size, and d) temperature. We aim to broaden the usefulness of nanoparticle targeting of pathogens with antibodies by 1) identifying design principles that offer a rational method for controlling the molecular affinity through simple modification of valency and 2) utilizing an antibody molecule that is heat-stable up to temperatures of 70¿C or greater. Successful completion of this proposal will open several fruitful paths of discovery which will result in improved sensors and countermeasures for pathogens and biowarfare agents. We anticipate the creation of nano/microparticles with tunable antigen affinity and heat stability. These two impacts will drastically improve the viability of antibody- based detectors, countermeasures, and diagnosis methods. PUBLIC HEALTH RELEVANCE: We propose to create nano/microparticles that are targeted by the newly discovered VLR antibody. We will create recombinant forms of VLRs that we anticipate will offer the following benefits to drug delivery, reagent development, and sensors, including rational adjustment of antibody affinity and temperature stability.