Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15.

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.

Cyclophosphamide-Mediated Tumor Priming for Enhanced Delivery and Antitumor Activity of HER2-Targeted Liposomal Doxorubicin (MM-302).

Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that effective tumor delivery and penetration are critical barriers to their clinical activity. HER2-targeted PEGylated liposomal doxorubicin (MM-302, HER2-tPLD) is an antibody-liposomal drug conjugate designed to deliver doxorubicin to HER2-overexpressing cancer cells while limiting uptake into nontarget cells. In this work, we demonstrate that the administration and appropriate dose sequencing of cyclophosphamide can improve subsequent MM-302 delivery and enhance antitumor activity in preclinical models without negatively affecting nontarget tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Furthermore, the effect was found to be unique to cyclophosphamide and related analogues, and not shared by other agents, such as taxanes or eribulin, under the conditions examined. Analysis of the cyclophosphamide-treated tumors suggests that the mechanism for improved MM-302 delivery involves the induction of tumor cell apoptosis, reduction of overall tumor cell density, substantial lowering of interstitial fluid pressure, and increasing vascular perfusion. The novel dosing strategy for cyclophosphamide described herein is readily translatable to standard clinical regimens, represents a potentially significant advance in addressing the drug delivery challenge, and may have broad applicability for nanomedicines. This work formed the basis for clinical evaluation of cyclophosphamide for improving liposome deposition as part of an ongoing phase I clinical trial of MM-302 in HER2-positive metastatic breast cancer.

A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)-as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.

Detection of ferrocenemethanol and molecular oxygen based on electrogenerated chemiluminescence quenching at a bipolar electrode.

Small molecules, such as ferrocenemethanol (FcMeOH) and O2, that are capable of quenching the Ru(bpy)3(2+) excited state via energy or electron transfer can be quantitatively detected in a bipolar electrochemical cell based on the attenuation of steady-state electrogenerated chemiluminescence (ECL). FcMeOH quenches ECL generated by the Ru(bpy)3(2+) oxalate coreactant system, exhibiting a linear dependence on [FcMeOH] with a Stern-Volmer slope of 921 M(-1), corresponding to a quenching rate constant of 2 × 10(9) M(-1) s(-1). We used the bipolar ECL quenching platform to measure dissolved O2 and validated the results using a standard Clark electrode. The detection limit for local [O2] measured using ECL quenching was found to be 300 ppb. This work opens up the possibility of utilizing ECL quenching at bipolar electrodes for a wide range of applications.

Quantitation of femtomolar protein levels via direct readout with the electrochemical proximity assay.

We have developed a separation-free, electrochemical assay format with direct readout that is amenable to highly sensitive and selective quantitation of a wide variety of target proteins. Our first generation of the electrochemical proximity assay (ECPA) is composed of two thrombin aptamers which form a cooperative complex only in the presence of target molecules, moving a methylene blue (MB)-conjugated oligonucleotide close to a gold electrode. Without washing steps, electrical current is increased in proportion to the concentration of a specific target protein. By employing a DNA-based experimental model with the aptamer system, we show that addition of a short DNA competitor can reduce background current of the MB peak to baseline levels. As such, the detection limit of aptamer-based ECPA for human thrombin was 50 pM via direct readout. The dual-probe nature of ECPA gave high selectivity and 93% recovery of signal from 2.5 nM thrombin in 2% bovine serum albumin (BSA). To greatly improve the flexibility of ECPA, we then proved the system functional with antibody-oligonucleotide conjugates as probes; the insulin detection limit was 128 fM with a dynamic range of over 4 orders of magnitude in concentration, again with high assay selectivity. ECPA thus allows separation-free, highly sensitive, and highly selective protein detection with a direct electrochemical readout. This method is extremely flexible, capable of detecting a wide variety of protein targets, and is amenable to point-of-care protein measurement, since any target with two aptamers or antibodies could be assayed via direct electrochemical readout.

Electrochemical sensors based on molecularly imprinted polymers grafted onto gold electrodes using click chemistry.

We have developed a three-step method to graft molecularly imprinted polymer (MIP) thin films onto Au electrodes. In the first step, propargyl acrylate is clicked onto an azidoundecanethiol (N(3)(CH(2))(11)SH)/decanethiol mixed self-assembled monolayer (SAM). Then, by applying UV light (365 nm) in the presence of N,N'-methylenebis(acrylamide) (MAAM) and azobisisobutyronitrile (AIBN) as the radical initiator, polymerization was carried out directly on the electrode surface in the presence of an electroactive template molecule, hydroquinone (HQ). Detection of HQ using the clicked-on MIP sensor was studied using chronoamperometry and its behavior was compared to that of a sensor prepared by drop-coating MIPs onto Au. The detection limit of the clicked-on MIP sensor for HQ was found to be 1.21±0.56 µM, about four times lower than what was observed using the coated-on MIP sensor. In addition, the sensitivity of the clicked-on MIP sensor was found to be approximately three times greater than the coated-on MIP sensor. Apparent diffusion coefficients determined using chronoamperometry suggest that the improved performance is likely due to the favorable mass transfer characteristics of the clicked-on MIP sensing membrane.

Screening the optical properties of Ag-Au alloy gradients formed by bipolar electrodeposition using surface enhanced Raman spectroscopy.

We report the synthesis of Ag-Au alloy gradients on stainless steel substrates using bipolar electrodeposition (BP-ED), a technique based on the existence of a potential gradient at the interface of a bipolar electrode (BPE) and an electrolytic solution. The interfacial potential gradient causes the rates of electrodeposition of Ag and Au to vary along the length of the BPE, leading to the electrodeposition of a chemical concentration gradient. The surface morphology of the electrodeposits was characterized using scanning electron microscopy (SEM), and their chemical composition was determined using energy dispersive X-ray spectroscopy (EDX). Self-assembled monolayers of a Raman-active probe molecule (benzene thiol) were allowed to form on the surface of the alloy gradients, and confocal Raman microscopy was employed to determine the alloy composition that resulted in the maximum surface enhanced Raman scattering (SERS) intensity. An alloy composition of ca. 70% Ag/30% Au was found to be optimum for SERS excited using 514.5 nm radiation, and it is explained on the basis of composition-dependent changes in the local surface plasmon resonance (LSPR) of the electrodeposited Ag-Au alloy.

Display of solid-state materials using bipolar electrochemistry.

We report the formation and characterization of one-dimensional chemical composition gradients of CdS on Au surfaces using bipolar electrodeposition. When an external electric field is applied across an electrically floating Au electrode immersed in a bipolar electrochemical cell, a position-dependent interfacial potential difference is generated along the length of the Au. This potential gradient can be used to induce variations of chemical composition within thin films electrodeposited onto the Au bipolar electrode (BPE). Thin films formed by bipolar electrodeposition represent continuous one-dimensional solid-state material libraries and were screened using resonance Raman microscopy and Auger electron spectroscopy. As predicted from simple thermodynamic considerations, we observed three distinct deposition zones scanning from the cathodic pole to the midpoint of the BPE: (i) CdS+Cd, (ii) stoichiometric CdS, and (iii) elemental S. Bipolar electrodeposition can be used to generate material libraries rapidly and without direct electrical contact to the substrate using extremely simple instrumentation.

Synthesis of metal-semiconductor core-shell nanoparticles using electrochemical surface-limited reactions.

We report the synthesis of Au/CuI and Au/CdS core-shell nanoparticle (NP) thin films using codeposition and electrochemical atomic layer deposition (EC-ALD). Au nanoparticle films were prepared on glassy carbon supports by depositing alternating layers of poly(diallyl dimethylammonium)-stabilized Au nanoparticles and CoP(2)W(17)O(61)(8-) polyoxometallate interlayers. From there, CuI was deposited onto the surface of Au nanoparticles using electrochemical atomic layer deposition, while CdS films were grown by an atom-by-atom codeposition method. The semiconductor-Au core-shell nanoparticles were characterized by electrochemistry, photoluminescence spectroscopy, and Raman spectroscopy. Our results indicate that the semiconductors deposit onto the AuNP surface by surface limited electrochemical reactions.

Preparation and characterization of polyoxometalate/protein ultrathin films grown on electrode surfaces using layer-by-layer assembly.

We report a new electrostatic layer-by-layer assembly method for the controlled deposition of electrocatalytically active enzymes onto electrode surfaces using polyoxometalate as the counteranion. Cytochrome c (cyt c), a redox active protein, and P(2)W(18)O(62)(6-), a Dawson-type polyoxometalate, were deposited onto glassy carbon electrodes by two procedures: static dipping and electrochemical cycling. Cyclic voltammetry and UV-vis spectroscopy reveal that approximately 1.5 x 10(-10) mol/cm(2) of P(2)W(18)O(62)(6-) and 2.2 x 10(-11) mol/cm(2) of cytochrome c are deposited per cycle, which correspond to approximately one monolayer of each molecule. The thicknesses of the resulting films measured by atomic force microscopy also indicate that the films are formed in a layer-by-layer fashion. Experimental factors that affect electron-transfer rate in these films, such as scan rate and film thickness, were systematically analyzed. The use of {P(2)W(18)O(62)(6-)/cyt c}n films to catalyze hydrogen peroxide reduction was demonstrated.

Improved oxygen reduction cathodes using polyoxometalate cocatalysts.

We report on the influence of a series of transition-metal-substituted Wells-Dawson (P2W17MnO62(12-n)-; M = WVI, FeII, CoII, RuII) and Keggin (PW12O40(3-) and PCoW11O39(5-) anions on the oxygen reduction reaction (ORR) at Au, Pd, and Pt. Wells-Dawson POMs adsorbed on Au lead to large positive shifts of the ORR potential. The magnitude of the shift depends on the transition metal and is explained using a simple thermodynamic model. The best cathode performance was achieved using a PCoW11O39(5-) cocatalyst and a Pt cathode. The +54 mV positive shift in the ORR potential that we observed is comparable to the performance of the best-known bimetallic catalysts.

Electron-acceptor-induced isomerization of aryl [6,5] open fulleroids to [6,6] closed methanofullerenes and the electrochemical evaluation of their free energy difference.

The thermal and photochemical rearrangements of a series of aryl substituted [6,5] open fulleroids to [6,6] closed methanofullerenes are accelerated in the presence in of electron acceptors. These [6,5] open fulleroids, facilitated by electron acceptors, rearrange thermally by a zwitteronic-type intermediate, while the photochemical reactions proceed via an excited-state electron-transfer process. The oxidation potentials of these [6,5] open fulleroids and their corresponding [6,6] closed methanofullerenes isomers have been evaluated. The free energy difference between the [6,5] open fulleroids and their corresponding [6,6] closed isomers have been estimated from the difference in their oxidation potentials.

A diruthenium-substituted polyoxometalate as an electrocatalyst for oxygen generation.

Transition metal heteropolyanions have been used to catalyze a variety of organic oxidations but have not previously been used for O2 generation, despite sharing some structural similarities with dioxoruthenium water-oxidation catalysts. In this study, we report that the di-Ru-substituted polyoxometalate (POM) [Ru2Zn2(H2O)2(ZnW9O34)2]14- can be used to catalyze the electrochemical generation of O2. By comparing the behavior of this compound to that observed using a mono-Ru-substituted POM catalyst, we show that adjacent Ru sites are necessary to observe O2 generation. These observations suggest a reaction pathway involving two Ru-bound oxygen species combining to form O2 and are consistent with the accepted mechanism of electrochemical oxygen evolution. Finally, analysis of the observed electrode kinetics yields a Tafel slope of roughly 120 mV, which is similar to values reported previously for perovskite anodes.