Laura E. Ruff

Multivalent Porous Silicon Nanoparticles Enhance the Immune Activation Potency of Agonistic CD40 Antibody

One of the fundamental paradigms in the use of nanoparticles to treat disease is to evade or suppress the immune system in order to minimize systemic side effects and deliver sufficient nanoparticle quantities to the intended tissues. However, the immune system is the bodys most important and effective defense against diseases. It protects the host by identifying and eliminating foreign pathogens as well as self-malignancies. Here we report a nanoparticle engineered to work with the immune system, enhancing the intended activation of antigen presenting cells (APCs). We show that luminescent porous silicon nanoparticles (LPSiNPs), each containing multiple copies of an agonistic antibody (FGK45) to the APC receptor CD40, greatly enhance activation of B cells. The cellular response to the nanoparticle-based stimulators is equivalent to a 30-40 fold larger concentration of free FGK45. The intrinsic near-infrared photoluminescence of LPSiNPs is used to monitor degradation and track the nanoparticles inside APCs.

Targeted and reversible cancer cell-binding DNA nanoparticles

Abstract DNA nanoparticles (DeNAno) produced by rolling circle replication of circular oligonucleotide templates are a novel format for the selection of cell-binding reagents from randomized libraries. DeNAno particles consist of several hundred concatemers and can leverage that multivalency to bind to complex surfaces such as cells. In this study, an iterative bio-panning approach was used to recover particles that bound to the mouse pancreatic cancer line, Panc-02. These particles shared a primary sequence motif. Hybridization of a locked nucleic acid complimentary to this motif both inhibited cell binding and released pre-bound DeNAno particles from the cells. The monomeric form of one of the selected sequences was unable to compete with the cognate particle, consistent with a low-affinity, but high-avidity, type interaction. DeNAno library selection against cancer or other cell types can, thus, yield novel cell binding agents without a priori knowledge of particular cell surface molecules.

Selection of DNA nanoparticles with preferential binding to aggregated protein target

Abstract High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, lifes own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions.

Identification of Peptide Mimotope Ligands for Natalizumab

Mimotope peptides selected from combinatorial peptide libraries can be used as capture reagents for immunoassay detection of therapeutic monoclonal antibodies (mAbs). We report the use of phage display libraries to identify peptide ligands (VeritopesTM) that bind natalizumab, a therapeutic mAb indicated for use in multiple sclerosis. PKNPSKF is identified as a novel natalizumab-binding motif, and peptides containing this motif demonstrated utility as capture reagents in enzyme-linked immunosorbent assays (ELISAs). A peptide containing the identified motif was shown to be competitive with the natural ligand (α4-integrin) and a neutralizing anti-idiotype antibody for natalizumab binding, indicating that VeritopesTM act as surrogate ligands that bind the antigen binding site of natalizumab. Affinity maturation further confirmed the motif sequence and yielded peptides with greater apparent affinity by ELISA. VeritopesTM are promising assay reagents for therapeutic drug level monitoring.

Individualized Dosing of Therapeutic Monoclonal Antibodies—a Changing Treatment Paradigm?

The introduction of monoclonal antibodies (mAbs) to the treatment of inflammatory bowel disease (IBD) was an important medical milestone. MAbs have been demonstrated as safe and efficacious treatments of IBD. However, a large percentage of patients either fail to respond initially or lose response to therapy after a period of treatment. Although there are factors associated with poor treatment outcomes in IBD, one cause for treatment failure may be low mAb exposure. Consequently, gastroenterologists have begun using therapeutic drug monitoring (TDM) to guide dose adjustment. However, while beneficial, TDM does not provide sufficient information to effectively adjust doses. The pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs are complex, with numerous factors impacting on mAb PK and PD. The concept of dashboard-guided dosing based on Bayesian PK models allows physicians to combine TDM with factors influencing mAb PK to individualize therapy more effectively. One issue with TDM has been the slow turnaround of assay results, either necessitating an additional clinic visit for a sample or reacting to TDM results at a subsequent, rather than the current, dose. New point-of-care (POC) assays for mAbs are being developed that would potentially allow physicians to determine drug concentration quickly. However, work remains to understand how to determine what target exposure is needed for an individual patient, and whether the combination of POC assays and dashboards presents a safe approach with substantial outcome benefit over the current standard of care.

Combining Isothermal Amplification Techniques: Coupled RCA-LAMP

Loop-mediated isothermal amplification (LAMP) is a low-cost method of quasi-exponential DNA amplification which utilizes constant temperature and stem-loop structures formed during amplification to generate strong amplified signal in a short amount of time. Rolling circle amplification (RCA) is a technique for linear isothermal DNA amplification of circularized oligonucleotide templates. When combined with padlock probes, RCA can be used as a diagnostic assay with single-base specificity. Here, a process is described called RCA-LAMP, in which LAMP is coupled with RCA of padlock probes to combine the benefit of the specificity of padlock probes with the speed of the LAMP reaction. It was found that the RCA-LAMP reaction proceeded significantly faster than the comparable hyperbranched RCA (HRCA) reaction, and it could detect 100 circular probes in less than 1 h.

DeNAno: A Novel Multivalent Affinity Reagent Produced by Selection of RCA-Generated DNA Nanoparticle Libraries

Biomolecular recognition with strong binding of interacting partners plays an essential role in research, diagnostic, and therapeutic applications. Currently available tools—namely antibodies and aptamers—function through high affinity, limited valency interactions with their targets. DNA nanoparticles, termed DeNAno, are a novel molecular recognition platform that utilizes low-affinity multivalent interactions to enable specific binding to a target. DeNAno particles are produced by rolling circle amplification (RCA) of randomized circular oligonucleotide templates yielding concatemeric single-stranded DNAs that contain multiple copies of the template sequences and form compact nanoparticles under physiological conditions. Highly diverse libraries can be created when random sequences are inserted into the circular templates, and the resulting DNA nanoparticles encode unique secondary and tertiary structures that can be screened to identify those particles that strongly bind targets of interest, such as cells or protein-coated beads. Unlike traditional binding reagents, DeNAno particle interactions are not governed by high-affinity binding since the presence of many copies of identical sequence allows avidity to compensate when the target is multimeric one.