Kris Barreto

Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein‐Ligase System

Efforts to engineer recombinant antibodies for specific diagnostic and therapy applications are time consuming and expensive, as each new recombinant antibody needs to be optimized for expression, stability, bio‐distribution, and pharmacokinetics. We have developed a new way to construct recombinant antibody‐like “devices” by using a bottom‐up approach to build them from well‐behaved discrete recombinant antibody domains or “parts”. Studies on antibody structure and function have identified antibody constant and variable domains with specific functions that can be expressed in isolation. We used the SpyTag/SpyCatcher protein ligase to join these parts together, thereby creating devices with desired properties based on summed properties of parts and in configurations that cannot be obtained by using genetic engineering. This strategy will create optimized recombinant antibody devices at reduced costs and with shortened development times.

A Single-Framework Synthetic Antibody Library Containing a Combination of Canonical and Variable Complementarity-Determining Regions

Synthetic antibody libraries have been used to generate antibodies with favorable biophysical and pharmacological properties. Here, we describe the design, construction, and validation of a phage‐displayed antigen‐binding fragment (Fab) library built on a modified trastuzumab framework with four fixed and two diversified complementarity‐determining regions (CDRs). CDRs L1, L2, H1, and H2 were fixed to preserve the most commonly observed “canonical” CDR conformation preferred by the modified trastuzumab Fab framework. The library diversity was engineered within CDRs L3 and H3 by use of custom‐designed trinucleotide phosphoramidite mixes and biased towards human antibody CDR sequences. The library contained ≈7.6 billion unique Fabs, and >95 % of the library correctly encoded both diversified CDR sequences. We used this library to conduct selections against the human epidermal growth factor receptor‐3 extracellular domain (HER3‐ECD) and compared the CDR diversity of the naïve library and the anti‐HER3 selection pool by use of next‐generation sequencing. The most commonly observed CDR combination isolated, named Her3‐3, was overexpressed and purified in Fab and immunoglobulin G (IgG) formats. Fab HER3‐3 bound to HER3‐ECD with a KD value of 2.14 nm and recognized cell‐surface HER3. Although HER3‐3 IgG bound to cell‐surface HER3, it did not inhibit the proliferation of HER3‐positive cells. Near‐infrared imaging showed that Fab HER3‐3 selectively accumulated in a murine HER3‐postive xenograft, thus providing a lead for the development of HER3 imaging probes.

Allosteric Lariat Peptide Inhibitors of Abl Kinase

Going against tradition: although most kinase inhibitors are ATP competitive, lariat peptides inhibit Abl kinase activity in an ATP-uncompetitive manner. Further, lariat peptides discriminated Src family kinases, and recognize the allosteric region that lies adjacent to the ATP binding pocket in the Abl kinase catalytic cleft.

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging

PurposeConstruction of antibody-based, molecular-targeted optical imaging probes requires the labeling of an antibody with a fluorophore. The most common method for doing this involves non-specifically conjugating a fluorophore to an antibody, resulting in poorly defined, heterogeneous imaging probes that often have suboptimal in vivo behavior. We tested a new strategy to site-specific label antibody-based imaging probes using the SpyCatcher/SpyTag protein ligase system.ProceduresWe used the SpyCatcher/SpyTag protein ligase system to site specifically label nimotuzumab, an anti-EGFR antibody and an anti-HER3 diabody. To prevent the labeling from interfering with antigen binding, we introduced the SpyTag and SpyCatcher at the C-terminus of the antibody and diabody, respectively. Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. Site-specific labeling of the antibody and diabody was performed in two steps. First, we labeled the SpyCatcher with IRDye800CW-Maleimide and the SpyTag with IRDye800CW-NHS. Second, we conjugated the IRDye800CW-SpyCatcher and the IRDye800CW-SpyTag to the antibody or diabody, respectively. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively.ResultsExpression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. Site-specifically IRDye800CW-labeled imaging probes bound to their immobilized targets, cells expressing these targets, and selectively accumulated in xenografts.ConclusionsThese results highlight the ease and utility of using the modular SpyTag/SpyCatcher protein ligase system for site-specific fluorescent labeling of protein-based imaging probes. Imaging probes labeled in this manner will be useful for optical imaging applications such as image-guided surgery and have broad application for other imaging modalities.