Design of synthetic recombinant antibody devices for applications in molecular targeted imaging

抗体体积大，具有可结晶片段(Fc)结构域，导致组织渗透率低，体内半衰期长，生产方法昂贵，有时还很困难，通常不适合许多应用。重组抗体(RAb)片段通过控制RAb的大小和构型、结合亲和力和特异性、Fc结构域特性以及通过将RAb与其他便于检测和纯化的蛋白质或标签融合来克服这些限制。这些Rab片段通常需要复杂的抗体结构，这可能导致低表达、较差的溶解性和聚集性以及毒性。因此，许多RAB片段需要昂贵和复杂的制造，必须针对每个RAB片段进行优化。 我们开发了一种新的方法来构建类Rab蛋白，使用具有离散的行为良好的抗体结构域或“部分”的“自下而上”合成策略。为此，我们使用蛋白质连接酶在不同的组合中表达和翻译后组装抗体恒定和可变域部分，从零开始创建具有所需性质的RAB样装置，这些性质基于各个部分的总和性质。我们使用SpyTag/Spycatcher连接酶系统在翻译后组装抗体部分和其他不同组合的蛋白质/肽结构域，其中一些在自然界或使用基因工程方法是不可能产生的。 我们研究计划的长期目标是开发一种高效、成本效益高的策略，利用离散的抗体结构域部件组装RAB类设备，用于基础科学和工程、诊断和治疗应用。为了实现这一目标，我们正在追求我们的短期目标，即设计、建造、提纯和验证用于分子靶向和预靶向成像的类RAB设备。我们建议建立一个抗体结构域部分的文库，这些部分可以使用SpyTag/Spycatcher蛋白连接酶系统在翻译后组装成复杂的设备。为了实现这一短期目标，我们已经确定了以下目标：(I)开发一个改进的噬菌体展示平台来产生可变域部分；(Ii)建立使用SpyTag/Spycatcher系统组装多价探针进行分子成像的方法；(Iii)改进体内SpyTag/Spycatcher连接；以及(Iv)验证多价Rabs用于分子靶向和预靶向成像。 这种用于RAB模块化组装的合成生物学方法将提高难以表达的RAB的产量，提供快速生产RAB的方法，并允许抗体结构域以使用基因工程方法无法获得的方式连接。使用易于生产和特性良好的抗体部分来构建RAB装置，将减少开发成本和时间，并提高RAB用于成像和治疗的安全性。

Antibodies are large in size and possess a crystallizable fragment (Fc) domain, resulting in low tissue penetration, long in vivo half lives, and expensive and sometimes difficult production methods, which are often not suitable for many applications. Recombinant antibody (rAb) fragments have been engineered to overcome these limitations by controlling rAb size and configurations, binding affinities and specificities, Fc domain properties, and by fusing rAbs to other proteins or tags that facilitate detection and purification. These rAb fragments often require sophisticated antibody architectures not present in nature, which can result in low expression, poor solubility and aggregation, and toxicity. Thus, many rAb fragments require expensive and complex manufacturing that must be optimized for each rAb fragment. We developed a novel approach to construct rAb-like proteins using a “bottom up” synthetic strategy with discrete well behaved antibody domain or “parts”. To do this, we express and post translationally assemble antibody constant and variable domain parts in different combinations using a protein ligase to create rAb like devices from scratch with desired properties that are based on the summed properties of the individual parts. We use the SpyTag/SpyCatcher ligase system to post-translationally assemble antibody parts and other protein/peptide domains in different combinations, some of which are not possible to generate in nature or using genetic engineering approaches. The long term objective of our research program is to develop an efficient, cost effective strategy for assembling rAb-like devices from discrete antibody domain parts for applications in basic science and engineering, diagnostics, and therapy. To achieve this, we are pursuing our short-term objective of designing, constructing, purifying, and validating rAb-like devices for molecular targeted and pre-targeted imaging. We propose to establish a library of antibody domain parts that can be assembled post-translationally into complex devices using the SpyTag/SpyCatcher protein ligase system. To achieve this short-term objective, we have established the following aims: (i) Develop an improved phage display platform for generating variable domain parts; (ii) Establish methods to use the SpyTag/SpyCatcher system to assemble multivalent probes for molecular imaging; (iii) Improve the in vivo SpyTag/SpyCatcher ligation; and (iv) Validate multivalent rAbs for molecular targeted and pretargeted imaging. This synthetic biology approach for the modular assembly of rAbs will improve yields of rAbs that are difficult to express, provide rapid methods to produce rAbs, and allow antibody domains to be connected in ways that cannot be obtained by using genetic engineering approaches. Using easy to produce and well characterized antibody parts to construct rAb devices will reduce development costs and time, and improve the safety of rAbs used in imaging and therapy.