Grant: Discovery Grants Program - Individual

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.