Post-translational Assembly of Protein Parts into Complex Devices by Using SpyTag/SpyCatcher Protein Ligase.

Exploiting the innate modularity of proteins has allowed advances across the fields of synthetic biology and biotechnology. By using standardized protein components as building blocks, complex, multiprotein assemblies with sophisticated functions can be generated; feats previously not possible with strictly genetic-engineering approaches. The development of strategies for protein assembly is accelerating, pushing the boundaries of protein architecture. SpyTag and SpyCatcher protein ligase is a recent advance in this field that allows plug-and-play modularity by harnessing post-translational protein assembly. Herein, we review the latest applications of this powerful tool including novel enzyme assemblies, modularizing protein display, and the generation of antibody and antibody-like "devices" by using SpyTag/SpyCatcher technology.

A novel synthetic trivalent single chain variable fragment (tri-scFv) construction platform based on the SpyTag/SpyCatcher protein ligase system.

BACKGROUND: Advances in antibody engineering provide strategies to construct recombinant antibody-like molecules with modified pharmacokinetic properties. Multermerization is one strategy that has been used to produce antibody-like molecules with two or more antigen binding sites. Multimerization enhances the functional affinity (avidity) and can be used to optimize size and pharmacokinetic properties. Most multimerization strategies involve genetically fusing or non-covalently linking antibody fragments using oligomerization domains. Recent studies have defined guidelines for producing antibody-like molecules with optimal tumor targeting properties, which require intermediates size (70-120 kDa) and bi- or tri-valency. RESULTS: We described a highly modular antibody-engineering platform for rapidly constructing synthetic, trivalent single chain variable fragments (Tri-scFv) using the SpyCatcher/SpyTag protein ligase system. We used this platform to construct an anti-human epidermal growth factor receptor 3 (HER3) Tri-scFv. We generated the anti-HER3 Tri-scFv by genetically fusing a SpyCatcher to the C-terminus of an anti-HER3 scFv and ligating it to a synthetic Tri-SpyTag peptide. The anti-HER3 Tri-scFv bound recombinant HER3 with an apparent KD of 2.67 nM, which is approximately 12 times lower than the KD of monomeric anti-HER3 scFv (31.2 nM). Anti-HER3 Tri-scFv also bound endogenous cell surface expressed HER3 stronger than the monomer anti-HER3 scFv. CONCLUSION: We used the SpyTag/SpyCatcher protein ligase system to ligate anti-HER3 scFv fused to a SpyCatcher at its C-termini to a Tri-SpyTag to construct Tr-scFv. This system allowed the construction of a Tri-scFv with all the scFv antigen-binding sites pointed outwards. The anti-HER3 Tri-scFv bound recombinant and endogenously expressed HER3 with higher functional affinity (avidity) than the monomeric anti-HER3 scFv. The Tri-scFv had the size, valency, and functional affinity that are desired for therapeutic and imaging applications. Use of the SpyTag/SpyCatcher protein ligase system allows Tri-scFvs to be rapidly constructed in a simple, modular manner, which can be easily applied to scFvs or other antibody fragments targeting other antigens.

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.

Do Islamic banks use institutional theory in the light of Shariah governance? Empirical evidence from a Muslim dominant country.

The present paper examines the application of institutional theory (hereafter IT) mechanisms (isomorphism and legitimacy) in Shariah governance (henceforth SG) practices and in operations, as it best illuminates the operational guidelines and procedures for the Islamic banks to attain legitimacy. More specifically, the study aims to empirically explore the SG of Islamic banks in Bangladesh from the viewpoint of IT mechanisms. The study applied a qualitative case study method and a semi-structured face-to-face (individual) interview with various stakeholders connected with Bangladesh's Islamic banks and SG practices. The study finds that Islamic banks face pressure from the administration, regulators, and government in performing their activities and functions. More importantly, most Islamic banks replicate one leading Islamic bank, which indicates the presence of mimetic or cultural-cognitive isomorphism. Conversely, normative pressure covers the existing practicing environment, people's social awareness, knowledge, willingness, and accountability of the Board of Directors (BOD) towards the people concerning Shariah compliance with policymaking. Moreover, socio-political norms, regulations, and corruption influenced policymaking, preparation of SG guidelines, and their implementation. The study significantly contributed to national regulatory bodies by exploring the presence of significant pressures on diverse stakeholders. The study has several policy contributions for the Islamic banks and the Central Bank of Bangladesh. Notably, the study will help global regulatory bodies and policymakers minimize multifaceted pressures, improve SG practices, and enhance the quality of Shariah compliance.

Aspergillus nidulans galactofuranose biosynthesis affects antifungal drug sensitivity.

The cell wall is essential for fungal survival in natural environments. Many fungal wall carbohydrates are absent from humans, so they are a promising source of antifungal drug targets. Galactofuranose (Galf) is a sugar that decorates certain carbohydrates and lipids. It comprises about 5% of the Aspergillus fumigatus cell wall, and may play a role in systemic aspergillosis. We are studying Aspergillus wall formation in the tractable model system, A. nidulans. Previously we showed single-gene deletions of three sequential A. nidulans Galf biosynthesis proteins each caused similar hyphal morphogenesis defects and 500-fold reduced colony growth and sporulation. Here, we generated ugeA, ugmA and ugtA strains controlled by the alcA(p) or niiA(p) regulatable promoters. For repression and expression, alcA(p)-regulated strains were grown on complete medium with glucose or threonine, whereas niiA(p)-regulated strains were grown on minimal medium with ammonium or nitrate. Expression was assessed by qPCR and colony phenotype. The alcA(p) and niiA(p) strains produced similar effects: colonies resembling wild type for gene expression, and resembling deletion strains for gene repression. Galf immunolocalization using the L10 monoclonal antibody showed that ugmA deletion and repression phenotypes correlated with loss of hyphal wall Galf. None of the gene manipulations affected itraconazole sensitivity, as expected. Deletion of any of ugmA, ugeA, ugtA, their repression by alcA(p) or niiA(p), OR, ugmA overexpression by alcA(p), increased sensitivity to Caspofungin. Strains with alcA(p)-mediated overexpression of ugeA and ugtA had lower caspofungin sensitivity. Galf appears to play an important role in A. nidulans growth and vigor.

Author Correction: Synthetic antigen-binding fragments (Fabs) against S. mutans and S. sobrinus inhibit caries formation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nimotuzumab Site-Specifically Labeled with 89Zr and 225Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with 89Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (225Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and 225Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to 225Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.

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

PURPOSE: Construction 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. PROCEDURES: We 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. RESULTS: 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. 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. CONCLUSIONS: These 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.

Synthetic antigen-binding fragments (Fabs) against S. mutans and S. sobrinus inhibit caries formation.

Streptococcus mutans and Streptococcus sobrinus are the main causative agents of human dental caries. Current strategies for treating caries are costly and do not completely eradicate them completely. Passive immunization using nonhuman antibodies against Streptococcal surface antigens has shown success in human trials, however they often invoke immune reactions. We used phage display to generate human antigen-binding fragments (Fabs) against S. mutans and S. sobrinus. These Fabs were readily expressed in E. coli and bound to the surface S. mutans and S. sobrinus. Fabs inhibited sucrose-induced S. mutans and S. sobrinus biofilm formation in vitro and a combination of S. mutans and S. sobrinus Fabs prevented dental caries formation in a rat caries model. These results demonstrated that S. mutans and S. sobrinus Fabs could be used in passive immunization strategies to prevent dental caries. In the future, this strategy may be applied towards a caries therapy, whereby Fabs are topically applied to the tooth surface.

RecA Inhibitors Potentiate Antibiotic Activity and Block Evolution of Antibiotic Resistance.

Antibiotic resistance arises from the maintenance of resistance mutations or genes acquired from the acquisition of adaptive de novo mutations or the transfer of resistance genes. Antibiotic resistance is acquired in response to antibiotic therapy by activating SOS-mediated DNA repair and mutagenesis and horizontal gene transfer pathways. Initiation of the SOS pathway promotes activation of RecA, inactivation of LexA repressor, and induction of SOS genes. Here, we have identified and characterized phthalocyanine tetrasulfonic acid RecA inhibitors that block antibiotic-induced activation of the SOS response. These inhibitors potentiate the activity of bactericidal antibiotics, including members of the quinolone, ß-lactam, and aminoglycoside families in both Gram-negative and Gram-positive bacteria. They reduce the ability of bacteria to acquire antibiotic resistance mutations and to transfer mobile genetic elements conferring resistance. This study highlights the advantage of including RecA inhibitors in bactericidal antibiotic therapies and provides a new strategy for prolonging antibiotic shelf life.

Aspergillus nidulans cell wall composition and function change in response to hosting several Aspergillus fumigatus UDP-galactopyranose mutase activity mutants.

Deletion or repression of Aspergillus nidulans ugmA (AnugmA), involved in galactofuranose biosynthesis, impairs growth and increases sensitivity to Caspofungin, a ß-1,3-glucan synthesis antagonist. The A. fumigatus UgmA (AfUgmA) crystal structure has been determined. From that study, AfUgmA mutants with altered enzyme activity were transformed into AnugmA▵ to assess their effect on growth and wall composition in A. nidulans. The complemented (AnugmA::wild type AfugmA) strain had wild type phenotype, indicating these genes had functional homology. Consistent with in vitro studies, AfUgmA residues R182 and R327 were important for its function in vivo, with even conservative amino (RK) substitutions producing AnugmA? phenotype strains. Similarly, the conserved AfUgmA loop III histidine (H63) was important for Galf generation: the H63N strain had a partially rescued phenotype compared to AnugmA▵. Collectively, A. nidulans strains that hosted mutated AfUgmA constructs with low enzyme activity showed increased hyphal surface adhesion as assessed by binding fluorescent latex beads. Consistent with previous qPCR results, immunofluorescence and ELISA indicated that AnugmA▵ and AfugmA-mutated A. nidulans strains had increased α-glucan and decreased ß-glucan in their cell walls compared to wild type and AfugmA-complemented strains. Like the AnugmA▵ strain, A. nidulans strains containing mutated AfugmA showed increased sensitivity to antifungal drugs, particularly Caspofungin. Reduced ß-glucan content was correlated with increased Caspofungin sensitivity. Aspergillus nidulans wall Galf, α-glucan, and ß-glucan content was correlated in A. nidulans hyphal walls, suggesting dynamic coordination between cell wall synthesis and cell wall integrity.