Nature-inspired design and evolution of anti-amyloid antibodies.

Antibodies that recognize amyloidogenic aggregates with high conformational and sequence specificity are important for detecting and potentially treating a wide range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, these types of antibodies are challenging to generate because of the large size, hydrophobicity, and heterogeneity of protein aggregates. To address this challenge, we developed a method for generating antibodies specific for amyloid aggregates. First, we grafted amyloidogenic peptide segments from the target polypeptide [Alzheimer's amyloid-ß (Aß) peptide] into the complementarity-determining regions (CDRs) of a stable antibody scaffold. Next, we diversified the grafted and neighboring CDR sites using focused mutagenesis to sample each WT or grafted residue, as well as one to five of the most commonly occurring amino acids at each site in human antibodies. Finally, we displayed these antibody libraries on the surface of yeast cells and selected antibodies that strongly recognize Aß-amyloid fibrils and only weakly recognize soluble Aß. We found that this approach enables the generation of monovalent and bivalent antibodies with nanomolar affinity for Aß fibrils. These antibodies display high conformational and sequence specificity as well as low levels of nonspecific binding and recognize a conformational epitope at the extreme N terminus of human Aß. We expect that this systematic approach will be useful for generating antibodies with conformational and sequence specificity against a wide range of peptide and protein aggregates associated with neurodegenerative disorders.

"Trim"ming PolyQ proteins with engineered PML.

Protein abnormalities are the major cause of neurodegenerative diseases such as spinocerebellar ataxia (SCA). Protein misfolding and impaired degradation leads to the build-up of protein aggregates inside the cell, which may further cause cellular degeneration. Reducing levels of either the soluble misfolded form of the protein or its precipitated aggregate, even marginally, could significantly improve cellular health. Despite numerous pre-existing strategies to target these protein aggregates, there is considerable room to improve their specificity and efficiency. In this study, we demonstrated the enhanced intracellular degradation of both monomers and aggregates of mutant ataxin1 (Atxn1 82Q) by engineering an E3 ubiquitin ligase enzyme, promyelocytic leukemia protein (PML). Specifically, we showed enhanced degradation of both soluble and aggregated Atxn1 82Q in mammalian cells by targeting this protein using PML fused to single chain variable fragments (scFvs) specific for monomers and aggregates of the target protein. The ability to solubilize Atxn1 82Q aggregates was due to the PML-mediated enhanced SUMOylation of the target protein. This ability to reduce the intracellular levels of both misfolded forms of Atxn1 82Q may not only be useful for treating SCA, but also applicable for the treatment of other PolyQ disorders.

Nanovaccines Displaying the Influenza Virus Hemagglutinin in an Inverted Orientation Elicit an Enhanced Stalk-Directed Antibody Response.

Despite the availability of licensed vaccines, influenza causes considerable morbidity and mortality worldwide. Current influenza vaccines elicit an immune response that primarily targets the head domain of the viral glycoprotein hemagglutinin (HA). Influenza viruses, however, readily evade this response by acquiring mutations in the head domain. While vaccines that target the more conserved HA stalk may circumvent this problem, low levels of antistalk antibodies are elicited by vaccination, possibly due to the poor accessibility of the stalk domain to B cell receptors. In this work, it is demonstrated that nanoparticles presenting HA in an inverted orientation generate tenfold higher antistalk antibody titers after a prime immunization and fivefold higher antistalk titers after a boost than nanoparticles displaying HA in its regular orientation. Moreover, nanoparticles presenting HA in an inverted orientation elicit a broader antistalk response that reduces mouse weight loss and improves survival after challenge to a greater extent than nanoparticles displaying HA in a regular orientation. Refocusing the antibody response toward conserved epitopes by controlling antigen orientation may enable the design of broadly protective nanovaccines targeting influenza viruses and other pathogens with pandemic potential.

The Design of Vaccines Based on the Shielding of Antigenic Site Ø of a Respiratory Syncytial Virus Fusion Protein Immunogen.

Respiratory syncytial virus (RSV), for which there is currently no licensed vaccine, displays a fusion (F) protein that is considered a vaccine target. This protein has an antigenic site called site Ø, which has been shown to elicit potent, neutralizing antibodies and has therefore been considered important in the formulation of RSV vaccines. However, this site is also the least conserved region on the F protein across RSV subtypes. Therefore, directing the immune response away from site Ø and refocusing it toward more conserved parts of the RSV F protein might serve to better elicit broadly neutralizing antibodies. To demonstrate that directing the immune response away from site Ø is a viable approach, a prefusion F-based vaccine based on an F protein with a shielded site Ø is generated. Sera from mice immunized with multivalent scaffolds presenting this immunogen is capable of neutralizing RSV of both subtypes. This result may have application in the development of an effective and broadly protective RSV vaccine.

One-pot synthesis of heterodimeric agonists that activate the canonical Wnt signaling pathway.

Fragment antigen-binding domains (Fabs) from anti-Frizzled and anti-LRP6 monoclonal antibodies were conjugated using SpyTag-SpyCatcher chemistry via a one-pot reaction. The resulting synthetic heterodimeric agonist outperformed the natural ligand, Wnt-3a, in activating canonical Wnt signaling in mammalian cells. This approach should be broadly applicable to activate receptor-mediated cellular signaling.

Designing Multivalent Ligands to Control Biological Interactions: From Vaccines and Cellular Effectors to Targeted Drug Delivery.

Multivalent interactions in which multiple ligands on one object bind to multiple receptors on another are commonly found in natural biological systems. In addition, these interactions can lead to increased strength and selectivity when compared to the corresponding monovalent interaction. These attributes have also guided the design of synthetic multivalent ligands to control biological interactions. This review will highlight the recent literature describing the use of multivalent ligand display in the design of vaccines, immunomodulators, cell signaling effectors, and vehicles for targeted drug delivery.

Nanopatterning protein antigens to refocus the immune response.

Vaccines for many important diseases remain elusive, and those for others need to be updated frequently. Vaccine efficacy has been hindered by existing sequence diversity in proteins and by newly-acquired mutations that enable escape from vaccine-induced immune responses. To address these limitations, we developed an approach for nanopatterning protein antigens that combines the site-specific incorporation of non-canonical amino acids with chemical modification to focus the immune response on conserved protein regions. We demonstrated the approach using green fluorescent protein (GFP) as a model antigen and with a promising malaria vaccine candidate, Merozoite surface protein 119 (MSP119). Immunization of mice with nanopatterned MSP119 elicited antibodies that recognized MSP119 from heterologous strains, differing in sequence at as many as 21 of 96 residues. Nanopatterning should enable the elicitation of broadly protective antibodies against a wide range of pathogens and toxins.

Bionanotechnology for vaccine design.

There have been significant advances in the design of nanostructured scaffolds for eliciting robust immune responses. One method to produce strong immune responses is to emulate the appearance of a pathogen. Since pathogens such as viruses and bacteria often display multiple copies of ligands on their surfaces, the immune system is particularly sensitive towards multivalent displays of antigens. Consequently, when designing a vaccine, it is advantageous to decorate a nanostructured surface with multiple copies of an antigen. This review highlights the design and efficacy of a diverse set of recently developed nanostructured vaccine scaffolds.

Synthesis of multivalent polymer-aptamer conjugates with enhanced inhibitory potency.

PURPOSE: We are interested in designing a modular strategy for creating potent multivalent ligands, which frequently can be used as effective inhibitors of undesired biomolecular interactions. For example, such inhibitors might prevent the self-assembly of bacterial toxins or the attachment of a virus to its host cell receptors. METHODS: We used a biocompatible polyamino acid polymer as a scaffold for grafting multiple copies of an oligonucleotide aptamer (OA). Specifically, the carboxylates on the side chains of polyglutamic acid (PGA) were modified with a thiol-reactive linker, N-aminoethyl maleimide (AEM), and thiol-functionalized OAs were attached to the maleimide moieties. The resulting conjugates were tested for their ability to compete with and inhibit the binding of unconjugated monovalent OAs to the target cell receptor. RESULTS: Multivalent PGA-OA conjugates with low, medium, and high valency were successfully prepared. The varying valency and successful purification to remove unconjugated OAs were confirmed by polyacrylamide gel electrophoresis. The resulting purified conjugates inhibited the binding of unconjugated monovalent OAs, and the measured half maximal inhibitory concentration (IC50) values corresponded to a 38-88-fold enhancement of potency on a per-aptamer basis, relative to OA alone. CONCLUSION: Multivalent conjugation of OA ligands has potential as a generally useful way to improve the potency of the interaction between the ligand and its target receptor. We have demonstrated this principle with a known OA as a proof of concept as well a synthetic strategy that can be used to synthesize multivalent conjugates of other OAs.

Tunable aggregation of gold-silica janus nanoparticles to enable contrast-enhanced multiwavelength photoacoustic imaging in vivo.

Photoacoustic imaging using exogenous contrast agents has emerged as a hybrid technique that enables the deep imaging of optical properties of tissues with high spatial resolution. The power of this imaging technique can be greatly enhanced by the use of contrast agents that absorb at near-infrared wavelengths and whose optical properties can be modulated in response to the local environment. We have designed contrast agents consisting of gold nanoparticles coated with anisotropic silica nanoshells. The tunable aggregation of these janus particles in cell culture media resulted in a dramatic amplification of photoacoustic signals in the near-infrared region. We also demonstrated imaging using these contrast agents in mammalian cells, including macrophages and breast cancer cells as well as in vivo. The ability to modulate janus particle aggregation in response to a range of stimuli in combination with the high resolution and deep penetration of multiwavelength photoacoustic imaging are attractive for a broad range of applications in diagnostic imaging and theranostics.

Recent advances in micro/nanoscale biomedical implants.

The medical device industry is growing at a very fast pace and has recorded great research activity over the past decade. The interdisciplinary nature of this field has made it possible for researchers to incorporate principles from various allied areas like pharmaceutics, bioengineering, biotechnology, chemistry, electronics, biophysics etc. to develop superior medical solutions, offering better prospects to the patient. Moreover, micro and nanotechnology have made it possible to positively affect at the sub-micron scales, the cellular processes initiated upon implantation. Literature is rife with findings on various implants and this review comprehensively summarizes the recent advances in micro/nanoscale implantable medical devices - particularly cardiovascular implants, neural implants, orthopedic and dental implants and other miscellaneous implants. Over the years, medical implants have metamorphosed from mere support providing devices to smart interventions participating positively in the healing process. We have highlighted the current research in each area emphasizing on the value addition provided by micro/nanoscale features, its course through the past and the future perspectives focusing on the unmet needs.

Nanocoatings on implantable medical devices.

The global medical device industry has experienced significant growth over the past 5 years. The surge of patent publications in the field bears testimony to this fact. The advent of nanotechnology has opened up newer unexplored vistas in the field of medical devices. This review summarizes patents employing the principles of nanotechnology in the formulation of coatings for implantable medical devices. Patents selected have at least one entity or structure with dimensions in the nanometer range, which results in a therapeutic value addition. The strategies reviewed pertain to tackling issues such as restenosis and thrombosis in addition to improving the overall acceptability of the implantable medical device, particularly those placed in the vasculature.