Artificial Targets: a versatile cell-free platform to characterize CAR T cell function in vitro.

Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have tremendous potential and proven clinical efficacy against a number of malignancies. Research and development are emerging to deepen the knowledge of CAR T cell efficacy and extend the therapeutic potential of this novel therapy. To this end, functional characterization of CAR T cells plays a central role in consecutive phases across fundamental research and therapeutic development, with increasing needs for standardization. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture with cell lines expressing the target antigen. However, the use of target cell lines poses several limitations, including alterations in cell fitness, metabolic state or genetic drift due to handling and culturing of the cells, which would increase variabilities and could lead to inconsistent results. Moreover, the use of target cell lines can be work and time intensive, and introduce significant background due to the allogenic responses of T cells. To overcome these limitations, we developed a synthetic bead-based platform ("Artificial Targets") to characterize CAR T cell function in vitro. These synthetic microparticles could specifically induce CAR T cell activation, as measured by CD69 and CD137 (4-1BB) upregulation. In addition, engagement with Artificial Targets resulted in induction of multiple effector functions of CAR T cells mimicking the response triggered by target cell lines including cytotoxic activity, as assessed by exposure of CD107a (LAMP-1), expression and secretion of cytokines, as well as cell proliferation. Importantly, in contrast to target cells, stimulation with Artificial Targets showed limited unspecific CAR T cell proliferation. Finally, Artificial Targets demonstrated flexibility to engage multiple costimulatory molecules that can synergistically enhance the CAR T cell function and represented a powerful tool for modulating CAR T cell responses. Collectively, our results show that Artificial Targets can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications.

Generation of Endotoxin-Specific Monoclonal Antibodies by Phage and Yeast Display for Capturing Endotoxin.

Endotoxin, a synonym for lipopolysaccharide (LPS), is anchored in the outer membranes of Gram-negative bacteria. Even minute amounts of LPS entering the circulatory system can have a lethal immunoactivating effect. Since LPS is omnipresent in the environment, it poses a great risk of contaminating any surface or solution, including research products and pharmaceuticals. Therefore, monitoring LPS contamination and taking preventive or decontamination measures to ensure human safety is of the utmost importance. Nevertheless, molecules used for endotoxin detection or inhibition often suffer from interferences, low specificity, and low affinity. For this reason, the selection of new binders that are biocompatible, easy to produce, and that can be used for biopharmaceutical applications, such as endotoxin removal, is of high interest. Powerful techniques for selecting LPS-binding molecules in vitro are display technologies. In this study, we established and compared the selection and production of LPS-specific, monoclonal, human single-chain variable fragments (scFvs) through two display methods: yeast and phage display. After selection, scFvs were fused to a human constant fragment crystallizable (Fc). To evaluate the applicability of the constructs, they were conjugated to polystyrene microbeads. Here, we focused on comparing the functionalized beads and their LPS removal capacity to a polyclonal anti-lipid A bead. Summarized, five different scFvs were selected through phage and yeast display, with binding properties comparable to a commercial polyclonal antibody. Two of the conjugated scFv-Fcs outperformed the polyclonal antibody in terms of the removal of LPS in aqueous solution, resulting in 265 times less residual LPS in solution, demonstrating the potential of display methods to generate LPS-specific binding molecules.

Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.

Tailored PGE2 Immunomodulation of moDCs by Nano-Encapsulated EP2/EP4 Antagonists.

Prostaglandin E2 (PGE2) is an important maturation mediator for dendritic cells (DCs). However, increased PGE2 levels in the tumor exert immunosuppressive effects on DCs by signaling through two E-Prostanoid (EP) receptors: EP2 and EP4. Blocking EP-receptor signaling of PGE2 with antagonists is currently being investigated for clinical applications to enhance anti-tumor immunity. In this study, we investigated a new delivery approach by encapsulating EP2/EP4 antagonists in polymeric nanoparticles. The nanoparticles were characterized for size, antagonist loading, and release. The efficacy of the encapsulated antagonists to block PGE2 signaling was analyzed using monocyte-derived DCs (moDCs). The obtained nanoparticles were sized between 210 and 260 nm. The encapsulation efficacy of the EP2/EP4 antagonists was 20% and 17%, respectively, and was further increased with the co-encapsulation of both antagonists. The treatment of moDCs with co-encapsulation EP2/EP4 antagonists prevented PGE2-induced co-stimulatory marker expression. Even though both antagonists showed a burst release within 15 min at 37 °C, the nanoparticles executed the immunomodulatory effects on moDCs. In summary, we demonstrate the functionality of EP2/EP4 antagonist-loaded nanoparticles to overcome PGE2 modulation of moDCs.

Pre-aggregation kinetics and intermediates of α-synuclein monitored by the ESIPT probe 7MFE.

The defining feature of the extensive family of amyloid diseases is the formation of networks of entangled elongated protein fibrils and amorphous aggregates exhibiting crossed ß-sheet secondary structure. The time course of amyloid conversion has been studied extensively in vitro with the proteins involved in the neurodegenerative pathology of Parkinson's disease (α-synuclein), Alzheimer's disease (Tau) and Huntington's disease (Huntingtin). Although much is known about the thermodynamics and kinetics of the transition from a soluble, intrinsically disordered monomer to the fibrillar end state, the putative oligomeric intermediates, currently considered to be the major initiators of cellular toxicity, are as yet poorly defined. We have detected and characterized amyloid precursors by monitoring AS aggregation with ESIPT (excited state intramolecular protein transfer) probes, one of which, 7MFE [7-(3-maleimido-N-propanamide)-2-(4-diethyaminophenyl)-3-hydroxychromone], is introduced here and compared with a related compound, 6MFC, used previously. A series of 140 spectra for sparsely labeled AS was acquired during the course of aggregation, and resolved into the relative contributions (spectra, intensities) of discrete molecular species including the monomeric, fibrillar, and ensemble of intermediate forms. Based on these findings, a kinetic scheme was devised to simulate progress curves as a function of key parameters. An essential feature of the model, one not previously invoked in schemes of amyloid aggregation, is the catalysis of molecular fuzziness by discrete colloidal nanoparticles arising spontaneously via monomer condensation upon exposure of AS to ≥ 37 °C.

ESIPT and FRET probes for monitoring nanoparticle polymer coating stability.

Coating strategies of inorganic nanoparticles (NPs) can provide properties unavailable to the NP core alone, such as targeting, specific sensing, and increased biocompatibility. Non-covalent amphiphilic NP capping polymers function via hydrophobic interactions with surface ligands and are extensively used to transfer NPs to aqueous media. For applications of coated NPs as actuators (sensors, markers, or for drug delivery) in a complex environment, such as biological systems, it is important to achieve a deep understanding of the factors affecting coating stability and behavior. We have designed a system that tests the coating stability of amphiphilic polymers through a simple fluorescent readout using either polarity sensing ESIPT (excited state intramolecular proton transfer) dyes or NP FRET (Förster resonance energy transfer). The stability of the coating was determined in response to changes in polarity, pH and ionic strength in the medium. Using the ESIPT system we observed linear changes in signal up to ∼20-25% v/v of co-solvent addition, constituting a break point. Based on such data, we propose a model for coating instability and the important adjustable parameters, such as the electrical charge distribution. FRET data provided confirmatory evidence for the model. The ESIPT dyes and FRET based methods represent new, simple tools for testing NP coating stability in complex environments.

Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract.

Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.

Influence of gold nanoparticles on the kinetics of α-synuclein aggregation.

α-synuclein (AS) is a small (140 amino acids), abundant presynaptic protein, which lacks a unique secondary structure in aqueous solution. Amyloid aggregates of AS in dopaminergic neurons of the midbrain are the hallmark of Parkinson's disease (PD). The process of aggregation involves a series of complex structural transitions from innocuous monomeric AS to oligomeric, presumably neurotoxic, forms and finally to fibril formation. Despite its potential importance for understanding PD pathobiology and devising rational, targeted therapeutic strategies, the details of the aggregation process remain largely unknown. Methodologies and reagents capable of controlling the aggregation kinetics are essential tools for the investigation of the molecular mechanisms of amyloid diseases. In this work, we investigated the influence of citrate-capped gold nanoparticles on the aggregation kinetics of AS using a fluorescent probe (MFC) sensitive to the polarity of the molecular microenvironment via excited state intramolecular proton transfer (ESIPT). The particular effects on the half time, nucleation time, and growth rate were ascertained. Gold nanoparticles produced a strong acceleration of protein aggregation with an influence on both the nucleation and growth phases of the overall mechanism. The effects were dependent on the size and concentration of the nanoparticles, being strongest for nanoparticles 10 nm in diameter, which produced a 3-fold increase in the overall aggregation rate at concentrations as low as 20 nM.

Voltamperometric discrimination of urea and melamine adulterated skimmed milk powder.

Nitrogen compounds like urea and melamine are known to be commonly used for milk adulteration resulting in undesired intoxication; a well-known example is the Chinese episode occurred in 2008. The development of a rapid, reliable and economic test is of relevance in order to improve adulterated milk identification. Cyclic voltammetry studies using an Au working electrode were performed on adulterated and non-adulterated milk samples from different independent manufacturers. Voltammetric data and their first derivative were subjected to functional principal component analysis (f-PCA) and correctly classified by the KNN classifier. The adulterated and non-adulterated milk samples showed significant differences. Best results of prediction were obtained with first derivative data. Detection limits in milk samples adulterated with 1% of its total nitrogen derived from melamine or urea were as low as 85.0 mg · L(-1) and 121.4 mg · L(-1), respectively. We present this method as a fast and robust screening method for milk adulteration analysis and prevention of food intoxication.

Biophysical properties and cellular toxicity of covalent crosslinked oligomers of α-synuclein formed by photoinduced side-chain tyrosyl radicals.

Alpha-synuclein (αS), a 140 amino acid presynaptic protein, is the major component of the fibrillar aggregates (Lewy bodies) observed in dopaminergic neurons of patients affected by Parkinson's disease. It is currently believed that noncovalent oligomeric forms of αS, arising as intermediates in its aggregation, may constitute the major neurotoxic species. However, attempts to isolate and characterize such oligomers in vitro, and even more so in living cells, have been hampered by their transient nature, low concentration, polymorphism, and inherent instability. In this work, we describe the preparation and characterization of low molecular weight covalently bound oligomeric species of αS obtained by crosslinking via tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine in the presence of ammonium persulfate. Numerous analytical techniques were used to characterize the αS oligomers: biochemical (anion-exchange chromatography, SDS-PAGE, and Western blotting); spectroscopic (optical: UV/Vis absorption, steady state, dynamic fluorescence, and dynamic light scattering); mass spectrometry; and electrochemical. Light-controlled protein oligomerization was mediated by formation of Tyr-Tyr (dityrosine) dimers through -C-C- bonds acting as covalent bridges, with a predominant involvement of residue Y39. The diverse oligomeric species exhibited a direct effect on the in vitro aggregation behavior of wild-type monomeric αS, decreasing the total yield of amyloid fibrils in aggregation assays monitored by thioflavin T (ThioT) fluorescence and light scattering, and by atomic force microscopy (AFM). Compared to the unmodified monomer, the photoinduced covalent oligomeric species demonstrated increased toxic effects on differentiated neuronal-like SH-SY5Y cells. The results highlight the importance of protein modification induced by oxidative stress in the initial molecular events leading to Parkinson's disease.

Supramolecular non-amyloid intermediates in the early stages of α-synuclein aggregation.

The aggregation of α-synuclein is associated with progression of Parkinson's disease. We have identified submicrometer supramolecular structures that mediate the early stages of the overall mechanism. The sequence of structural transformations between metastable intermediates were captured and characterized by atomic force microscopy guided by a fluorescent probe sensitive to preamyloid species. A novel ~0.3-0.6 µm molecular assembly, denoted the acuna, nucleates, expands, and liberates fibers with distinctive segmentation and a filamentous fuzzy fringe. These fuzzy fibers serve as precursors of mature amyloid fibrils. Cryo-electron tomography resolved the acuna inner structure as a scaffold of highly condensed colloidal masses interlinked by thin beaded threads, which were perceived as fuzziness by atomic force microscopy. On the basis of the combined data, we propose a sequential mechanism comprising molecular, colloidal, and fibrillar stages linked by reactions with disparate temperature dependencies and distinct supramolecular forms. We anticipate novel diagnostic and therapeutic approaches to Parkinson's and related neurodegenerative diseases based on these new insights into the aggregation mechanism of α-synuclein and intermediates, some of which may act to cause and/or reinforce neurotoxicity.

Highly Solvatochromic 7-Aryl-3-hydroxychromones.

Introduction of the dialkylaminophenyl group in position 7 of 3-hydroxychromone changes the orientation of the excited-state dipole moment and leads to superior solvatochromic properties (>170 nm emission shift in aprotic media). The excited-state intramolecular proton-transfer (ESIPT) reaction of 7-aryl-3-hydroxychromones is almost completely inhibited in most solvents. Methylation of the 3-OH abolishes ESIPT completely and also leads to improved photostability. The probes exhibit a ∼100-fold increase in fluorescence intensity and large Stokes shifts upon binding to membranes, reflecting differences in membrane phase and charge by a >40 nm spread in the emission band position.

Fluorescent ratiometric MFC probe sensitive to early stages of alpha-synuclein aggregation.

We introduce a sensor molecule, AS140-MFC, consisting of a covalent adduct of an Ala-to-Cys mutant of alpha-synuclein with the 3-hydroxychromone dual emission dye MFC. We show that the AS140-MFC construct is a multiparametric fluorescent probe suitable for the continuous monitoring of protein aggregation and is sensitive to the early and intermediate stages of alpha-synuclein aggregation, a process associated with Parkinson's disease.