Design of Crosslinking Antibodies For T-Cell Activation: Experimental and Computational Analysis of PD-1/CD137 Bispecific Agents.

Bispecific and multispecific agents have become increasingly utilized in cancer treatment and immunotherapy, yet their complex design parameters present a challenge in developing successful therapeutics. Bispecifics that crosslink receptors on two opposing cells can provide specific activation of a receptor only when these cells are in close spatial proximity, such as an immune cell and cancer cell in a tumor. These agents, including T cell activating bispecifics, can avoid off-tumor toxicity through activation only in the tumor microenvironment by utilizing a tumor target to cluster T-cell receptors for a selective costimulatory signal. Here, we investigate a panel of PD-1/CD137 targeted Humabody VH domains to determine the key factors for T cell activation, such as affinity, valency, expression level, domain orientation, and epitope location. Target expression is a dominant factor determining both specificity and potency of T cell activation. Given an intrinsic expression level, the affinity can be tuned to modulate the level of activation and IC50 and achieve specificity between low and high expression levels. Changing the epitope location and linker length showed minor improvements to activation at low expression levels, but increasing the valency for the target decreased activation at all expression levels. By combining non-overlapping epitopes for the target, we achieved higher receptor activation at low expression levels. A kinetic model was able to capture these trends, offering support for the mechanistic interpretation. This work provides a framework to quantify factors for T cell activation by cell-crosslinking bispecific agents and guiding principles for the design of new agents.

Electrical Conductivity of Subsurface Ocean Analogue Solutions from Molecular Dynamics Simulations.

Investigating the habitability of ocean worlds is a priority of current and future NASA missions. The Europa Clipper mission will conduct approximately 50 flybys of Jupiter's moon Europa, returning a detailed portrait of its interior from the synthesis of data from its instrument suite. The magnetometer on board has the capability of decoupling Europa's induced magnetic field to high precision, and when these data are inverted, the electrical conductivity profile from the electrically conducting subsurface salty ocean may be constrained. To optimize the interpretation of magnetic induction data near ocean worlds and constrain salinity from electrical conductivity, accurate laboratory electrical conductivity data are needed under the conditions expected in their subsurface oceans. At the high-pressure, low-temperature (HPLT) conditions of icy worlds, comprehensive conductivity data sets are sparse or absent from either laboratory data or simulations. We conducted molecular dynamics simulations of candidate ocean compositions of aqueous NaCl under HPLT conditions at multiple concentrations. Our results predict electrical conductivity as a function of temperature, pressure, and composition, showing a decrease in conductivity as the pressure increases deeper into the interior of an icy moon. These data can guide laboratory experiments at conditions relevant to icy moons and can be used in tandem to forward-model the magnetic induction signals at ocean worlds and compare with future spacecraft data. We discuss implications for the Europa Clipper mission.

Impact of tissue penetration and albumin binding on design of T cell targeted bispecific agents.

Bispecific agents are a rapidly growing class of cancer therapeutics, and immune targeted bispecific agents have the potential to expand functionality well beyond monoclonal antibody agents. Humabodies⁎ are fully human single domain antibodies that can be linked in a modular fashion to form multispecific therapeutics. However, the effect of heterogeneous delivery on the efficacy of crosslinking bispecific agents is currently unclear. In this work, we utilize a PSMA-CD137 Humabody with an albumin binding half-life extension (HLE) domain to determine the impact of tissue penetration on T cell activating bispecific agents. Using heterotypic spheroids, we demonstrate that increased tissue penetration results in higher T cell activation at sub-saturating concentrations. Next, we tested the effect of two different albumin binding moieties on tissue distribution using albumin-specific HLE domains with varying affinities for albumin and a non-specific lipophilic dye. The results show that a specific binding mechanism to albumin does not influence tissue penetration, but a non-specific mechanism reduced both spheroid uptake and distribution in the presence of albumin. These results highlight the potential importance of tissue penetration on bispecific agent efficacy and describe how the design parameters including albumin-binding domains can be selected to maximize the efficacy of bispecific agents.

Investigating Europa's Habitability with the Europa Clipper.

The habitability of Europa is a property within a system, which is driven by a multitude of physical and chemical processes and is defined by many interdependent parameters, so that its full characterization requires collaborative investigation. To explore Europa as an integrated system to yield a complete picture of its habitability, the Europa Clipper mission has three primary science objectives: (1) characterize the ice shell and ocean including their heterogeneity, properties, and the nature of surface-ice-ocean exchange; (2) characterize Europa's composition including any non-ice materials on the surface and in the atmosphere, and any carbon-containing compounds; and (3) characterize Europa's geology including surface features and localities of high science interest. The mission will also address several cross-cutting science topics including the search for any current or recent activity in the form of thermal anomalies and plumes, performing geodetic and radiation measurements, and assessing high-resolution, co-located observations at select sites to provide reconnaissance for a potential future landed mission. Synthesizing the mission's science measurements, as well as incorporating remote observations by Earth-based observatories, the James Webb Space Telescope, and other space-based resources, to constrain Europa's habitability, is a complex task and is guided by the mission's Habitability Assessment Board (HAB).

Low-Temperature Specific Heat Capacity of Water-Ammonia Mixtures Down to the Eutectic.

Robust thermodynamic data are essential for the development of geodynamic and geochemical models of ocean worlds. The water-ammonia system is of interest in the study of ocean worlds due to its purported abundance in the outer solar system, geological implications, and potential importance for origins of life. In support of developing new equations of state, we conducted 1 bar specific heat capacity measurements (Cp) using a differential scanning calorimeter (DSC) at low temperatures (184-314 K) and low mass fractions of ammonia (5.2-26.9 wt %) to provide novel data in the parameter space most relevant for planetary studies. This is the first known set of data with sufficient fidelity to investigate the trend of specific heat capacity with respect to temperature. The obtained Cp in the liquid phase domain above the liquidus generally increases with temperature. Deviations of our data from the currently adopted equation of state by Tillner-Roth and Friend[Tillner-Roth R.; Friend D. G.J. Phys. Chem. Ref. Data1998, 27, 63-96]. are generally negative (ranging from +1 to -10%) and larger at lower temperatures. This result suggests that suppression of the critical behavior of supercooled water (rapid increase in specific heat with decreasing temperature) by ammonia starts at a smaller concentration than that set by Tillner-Roth and Friend.[Tillner-Roth R.; Friend D. G.J. Phys. Chem. Ref. Data1998, 27, 63-96]. Cp measurements of the liquid were also obtained in the partial melting domain between the eutectic and liquidus. This novel data set will be useful in future investigations of conditions where such partial melt may exist, such as the ice shell-ocean boundary or the interiors of ocean worlds that may contain relatively large proportions of dissolved ammonia.

On the identification of hyperhydrated sodium chloride hydrates, stable at icy moon conditions.

Sodium chloride is expected to be found on many of the surfaces of icy moons like Europa and Ganymede. However, spectral identification remains elusive as the known NaCl-bearing phases cannot match current observations, which require higher number of water of hydration. Working at relevant conditions for icy worlds, we report the characterization of three "hyperhydrated" sodium chloride (SC) hydrates, and refined two crystal structures [2NaCl·17H2O (SC8.5); NaCl·13H2O (SC13)]. We found that the dissociation of Na+ and Cl- ions within these crystal lattices allows for the high incorporation of water molecules and thus explain their hyperhydration. This finding suggests that a great diversity of hyperhydrated crystalline phases of common salts might be found at similar conditions. Thermodynamic constraints indicate that SC8.5 is stable at room pressure below 235 K, and it could be the most abundant NaCl hydrate on icy moon surfaces like Europa, Titan, Ganymede, Callisto, Enceladus, or Ceres. The finding of these hyperhydrated structures represents a major update to the H2O-NaCl phase diagram. These hyperhydrated structures provide an explanation for the mismatch between the remote observations of the surface of Europa and Ganymede and previously available data on NaCl solids. It also underlines the urgent need for mineralogical exploration and spectral data on hyperhydrates at relevant conditions to help future icy world exploration by space missions.

Duality of Nrf2 in iron-overload cardiomyopathy.

Cardiomyopathy deeply affects quality of life and mortality of patients with b-thalassemia or with transfusion-dependent myelodysplastic syndromes. Recently, a link between Nrf2 activity and iron metabolism has been reported in liver ironoverload murine models. Here, we studied C57B6 mice as healthy control and nuclear erythroid factor-2 knockout (Nrf2-/-) male mice aged 4 and 12 months. Eleven-month-old wild-type and Nrf2-/- mice were fed with either standard diet or a diet containing 2.5% carbonyl-iron (iron overload [IO]) for 4 weeks. We show that Nrf2-/- mice develop an age-dependent cardiomyopathy, characterized by severe oxidation, degradation of SERCA2A and iron accumulation. This was associated with local hepcidin expression and increased serum non-transferrin-bound iron, which promotes maladaptive cardiac remodeling and interstitial fibrosis related to overactivation of the TGF-b pathway. When mice were exposed to IO diet, the absence of Nrf2 was paradoxically protective against further heart iron accumulation. Indeed, the combination of prolonged oxidation and the burst induced by IO diet resulted in activation of the unfolded protein response (UPR) system, which in turn promotes hepcidin expression independently from heart iron accumulation. In the heart of Hbbth3/+ mice, a model of b-thalassemia intermedia, despite the activation of Nrf2 pathway, we found severe protein oxidation, activation of UPR system and cardiac fibrosis independently from heart iron content. We describe the dual role of Nrf2 when aging is combined with IO and its novel interrelation with UPR system to ensure cell survival. We open a new perspective for early and intense treatment of cardiomyopathy in patients with b-thalassemia before the appearance of heart iron accumulation.

Iron therapy mitigates chronic kidney disease progression by regulating intracellular iron status of kidney macrophages.

Systemic iron metabolism is disrupted in chronic kidney disease (CKD). However, little is known about local kidney iron homeostasis and its role in kidney fibrosis. Kidney-specific effects of iron therapy in CKD also remain elusive. Here, we elucidate the role of macrophage iron status in kidney fibrosis and demonstrate that it is a potential therapeutic target. In CKD, kidney macrophages exhibited depletion of labile iron pool (LIP) and induction of transferrin receptor 1, indicating intracellular iron deficiency. Low LIP in kidney macrophages was associated with their defective antioxidant response and proinflammatory polarization. Repletion of LIP in kidney macrophages through knockout of ferritin heavy chain (Fth1) reduced oxidative stress and mitigated fibrosis. Similar to Fth1 knockout, iron dextran therapy, through replenishing macrophage LIP, reduced oxidative stress, decreased the production of proinflammatory cytokines, and alleviated kidney fibrosis. Interestingly, iron markedly decreased TGF-ß expression and suppressed TGF-ß-driven fibrotic response of macrophages. Iron dextran therapy and FtH suppression had an additive protective effect against fibrosis. Adoptive transfer of iron-loaded macrophages alleviated kidney fibrosis, validating the protective effect of iron-replete macrophages in CKD. Thus, targeting intracellular iron deficiency of kidney macrophages in CKD can serve as a therapeutic opportunity to mitigate disease progression.

Science Objectives for Flagship-Class Mission Concepts for the Search for Evidence of Life at Enceladus.

Cassini revealed that Saturn's Moon Enceladus hosts a subsurface ocean that meets the accepted criteria for habitability with bio-essential elements and compounds, liquid water, and energy sources available in the environment. Whether these conditions are sufficiently abundant and collocated to support life remains unknown and cannot be determined from Cassini data. However, thanks to the plume of oceanic material emanating from Enceladus' south pole, a new mission to Enceladus could search for evidence of life without having to descend through kilometers of ice. In this article, we outline the science motivations for such a successor to Cassini, choosing the primary science goal to be determining whether Enceladus is inhabited and assuming a resource level equivalent to NASA's Flagship-class missions. We selected a set of potential biosignature measurements that are complementary and orthogonal to build a robust case for any life detection result. This result would be further informed by quantifications of the habitability of the environment through geochemical and geophysical investigations into the ocean and ice shell crust. This study demonstrates that Enceladus' plume offers an unparalleled opportunity for in situ exploration of an Ocean World and that the planetary science and astrobiology community is well equipped to take full advantage of it in the coming decades.

A perturbation method for evaluating the magnetic field induced from an arbitrary, asymmetric ocean world analytically.

Magnetic investigations of icy moons have provided some of the most compelling evidence available confirming the presence of subsurface, liquid water oceans. In the exploration of ocean moons, especially Europa, there is a need for mathematical models capable of predicting the magnetic fields induced under a variety of conditions, including in the case of asymmetric oceans. Existing models are limited to either spherical symmetry or assume an ocean with infinite conductivity. In this work, we use a perturbation method to derive a semi-analytic result capable of determining the induced magnetic moments for an arbitrary layered body, provided each layer is nearly spherical. Crucially, we find that degree-2 tidal deformation results in changes to the induced dipole moments. We demonstrate application of our results to models of plausible asymmetry from the literature within the oceans of Europa and Miranda and the ionospheres of Callisto and Triton. For the models we consider, we find that in the asymmetric case, the induced magnetic field differs by more than 2 nT near the surface of Europa, 0.25-0.5 nT at 1 R above Miranda and Triton, and is essentially unchanged for Callisto. For Miranda and Triton, this difference is as much as 20%-30% of the induced field magnitude. If measurements near the moons can be made precisely to better than a few tenths of a nT, these values may be used by future spacecraft investigations to characterize asymmetry within the interior of icy moons.

Underground Microseismic Event Monitoring and Localization within Sensor Networks.

Locating underground microseismic events is important for monitoring subsurface activity and understanding the planetary subsurface evolution. Due to bandwidth limitations, especially in applications involving planetarily-distributed sensor networks, networks should be designed to perform the localization algorithm in-situ, so that only the source location information needs to be sent out, not the raw data. In this paper, we propose a decentralized Gaussian beam time-reverse imaging (GB-TRI) algorithm that can be incorporated to the distributed sensors to detect and locate underground microseismic events with reduced usage of computational resources and communication bandwidth of the network. After the in-situ distributed computation, the final real-time location result is generated and delivered. We used a real-time simulation platform to test the performance of the system. We also evaluated the stability and accuracy of our proposed GB-TRI localization algorithm using extensive experiments and tests.

A Metamorphic Origin for Europa's Ocean.

Europa likely contains an iron-rich metal core. For it to have formed, temperatures within Europa reached ≳ 1250 K. Going up to that temperature, accreted chondritic minerals - for example, carbonates and phyllosilicates - would partially devolatilize. Here, we compute the amounts and compositions of exsolved volatiles. We find that volatiles released from the interior would have carried solutes, redox-sensitive species, and could have generated a carbonic ocean in excess of Europa's present-day hydrosphere, and potentially an early CO 2 atmosphere. No late delivery of cometary water was necessary. Contrasting with prior work, CO 2 could be the most abundant solute in the ocean, followed by Ca 2 + , SO 4 2 - , and HCO 3 - . However, gypsum precipitation going from the seafloor to the ice shell decreases the dissolved S/Cl ratio, such that Cl > S at the shallowest depths, consistent with recently inferred endogenous chlorides at Europa's surface. Gypsum would form a 3-10 km thick sedimentary layer at the seafloor.

Simulating Serpentinization as It Could Apply to the Emergence of Life Using the JPL Hydrothermal Reactor.

The molecules feeding life's emergence are thought to have been provided through the hydrothermal interactions of convecting carbonic ocean waters with minerals comprising the early Hadean oceanic crust. Few laboratory experiments have simulated ancient hydrothermal conditions to test this conjecture. We used the JPL hydrothermal flow reactor to investigate CO2 reduction in simulated ancient alkaline convective systems over 3 days (T = 120°C, P = 100 bar, pH = 11). H2-rich hydrothermal simulant and CO2-rich ocean simulant solutions were periodically driven in 4-h cycles through synthetic mafic and ultramafic substrates and Fe>Ni sulfides. The resulting reductants included micromoles of HS- and formate accompanied possibly by micromoles of acetate and intermittent minor bursts of methane as ascertained by isotopic labeling. The formate concentrations directly correlated with the CO2 input as well as with millimoles of Mg2+ ions, whereas the acetate did not. Also, tens of micromoles of methane were drawn continuously from the reactor materials during what appeared to be the onset of serpentinization. These results support the hypothesis that formate may have been delivered directly to a branch of an emerging acetyl coenzyme-A pathway, thus obviating the need for the very first hydrogenation of CO2 to be made in a hydrothermal mound. Another feed to early metabolism could have been methane, likely mostly leached from primary CH4 present in the original Hadean crust or emanating from the mantle. That a small volume of methane was produced sporadically from the 13CO2-feed, perhaps from transient occlusions, echoes the mixed results and interpretations from other laboratories. As serpentinization and hydrothermal leaching can occur wherever an ocean convects within anhydrous olivine- and sulfide-rich crust, these results may be generalized to other wet rocky planets and moons in our solar system and beyond.

Experimental and Simulation Efforts in the Astrobiological Exploration of Exooceans.

The icy satellites of Jupiter and Saturn are perhaps the most promising places in the Solar System regarding habitability. However, the potential habitable environments are hidden underneath km-thick ice shells. The discovery of Enceladus' plume by the Cassini mission has provided vital clues in our understanding of the processes occurring within the interior of exooceans. To interpret these data and to help configure instruments for future missions, controlled laboratory experiments and simulations are needed. This review aims to bring together studies and experimental designs from various scientific fields currently investigating the icy moons, including planetary sciences, chemistry, (micro-)biology, geology, glaciology, etc. This chapter provides an overview of successful in situ, in silico, and in vitro experiments, which explore different regions of interest on icy moons, i.e. a potential plume, surface, icy shell, water and brines, hydrothermal vents, and the rocky core.

Increased Tumor Penetration of Single-Domain Antibody-Drug Conjugates Improves In Vivo Efficacy in Prostate Cancer Models.

Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA-approved antibody-drug conjugates (ADC). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of prostate-specific membrane antigen-binding single-domain (Humabody) ADC constructs to demonstrate that tissue penetration of protein-drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower in vitro potency resulted in higher in vivo efficacy than other protein-drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin-binding domain to the single-domain ADCs. A construct lacking an albumin-binding domain was rapidly cleared, leading to lower tumor uptake (%ID/g) and decreased in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro potency alone for these protein-drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing. SIGNIFICANCE: A mechanistic study of protein-drug conjugates demonstrates that a lower potency compound is more effective in vivo than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution.

Self-Assembling Ice Membranes on Europa: Brinicle Properties, Field Examples, and Possible Energetic Systems in Icy Ocean Worlds.

Brinicles are self-assembling tubular ice membrane structures, centimeters to meters in length, found beneath sea ice in the polar regions of Earth. We discuss how the properties of brinicles make them of possible importance for chemistry in cold environments-including that of life's emergence-and we consider their formation in icy ocean worlds. We argue that the non-ice composition of the ice on Europa and Enceladus will vary spatially due to thermodynamic and mechanical properties that serve to separate and fractionate brines and solid materials. The specifics of the composition and dynamics of both the ice and the ocean in these worlds remain poorly constrained. We demonstrate through calculations using FREZCHEM that sulfate likely fractionates out of accreting ice in Europa and Enceladus, and thus that an exogenous origin of sulfate observed on Europa's surface need not preclude additional endogenous sulfate in Europa's ocean. We suggest that, like hydrothermal vents on Earth, brinicles in icy ocean worlds constitute ideal places where ecosystems of organisms might be found.

Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold.

Acyl carrier protein (ACP) domains act as interaction hubs within modular polyketide synthase (PKS) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular PKS that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACP segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACP topology in both cases. This article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional α-helix that behaves like a rigid component of the domain. The interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase PKS chain-extension modules. The polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be ~12 residues shorter than previously thought, imposing strict limits on ACP-mediated substrate delivery within and between PKS modules.