CYpHER: Catalytic extracellular targeted protein degradation with high potency and durable effect.

Many disease-causing proteins have multiple pathogenic mechanisms, and conventional inhibitors struggle to reliably disrupt more than one. Targeted protein degradation (TPD) can eliminate the protein, and thus all its functions, by directing a cell's protein turnover machinery towards it. Two established strategies either engage catalytic E3 ligases or drive uptake towards the endolysosomal pathway. Here we describe CYpHER (CatalYtic pH-dependent Endolysosomal delivery with Recycling) technology with potency and durability from a novel catalytic mechanism that shares the specificity and straightforward modular design of endolysosomal uptake. By bestowing pH-dependent release on the target engager and using the rapid-cycling transferrin receptor as the uptake receptor, CYpHER induces endolysosomal target delivery while re-using drug, potentially yielding increased potency and reduced off-target tissue exposure risks. The TfR-based approach allows targeting to tumors that overexpress this receptor and offers the potential for transport to the CNS. CYpHER function was demonstrated in vitro with EGFR and PD-L1, and in vivo with EGFR in a model of EGFR-driven non-small cell lung cancer.

Sensitive bispecific chimeric T cell receptors for cancer therapy.

The expression of a synthetic chimeric antigen receptor (CAR) to redirect antigen specificity of T cells is transforming the treatment of hematological malignancies and autoimmune diseases [1-7]. In cancer, durable efficacy is frequently limited by the escape of tumors that express low levels or lack the target antigen [8-12]. These clinical results emphasize the need for immune receptors that combine high sensitivity and multispecificity to improve outcomes. Current mono- and bispecific CARs do not faithfully recapitulate T cell receptor (TCR) function and require high antigen levels on tumor cells for recognition [13-17]. Here, we describe a novel synthetic chimeric TCR (ChTCR) that exhibits superior antigen sensitivity and is readily adapted for bispecific targeting. Bispecific ChTCRs mimic TCR structure, form classical immune synapses, and exhibit TCR-like proximal signaling. T cells expressing Bi-ChTCRs more effectively eliminated tumors with heterogeneous antigen expression in vivo compared to T cells expressing optimized bispecific CARs. The Bi-ChTCR architecture is resilient and can be designed to target multiple B cell lineage and multiple myeloma antigens. Our findings identify a broadly applicable approach for engineering T cells to target hematologic malignancies with heterogeneous antigen expression, thereby overcoming the most frequent mechanism of relapse after current CAR T therapies.

IKK2 controls the inflammatory potential of tissue-resident regulatory T cells in a murine gain of function model.

Loss-of-function mutations have provided crucial insights into the immunoregulatory actions of Foxp3+ regulatory T cells (Tregs). By contrast, we know very little about the consequences of defects that amplify aspects of Treg function or differentiation. Here we show that mice heterozygous for an Ikbkb gain-of-function mutation develop psoriasis. Doubling the gene dose (IkbkbGoF/GoF) results in dactylitis, spondylitis, and characteristic nail changes, which are features of psoriatic arthritis. IkbkbGoF mice exhibit a selective expansion of Foxp3 + CD25+ Tregs of which a subset express IL-17. These modified Tregs are enriched in both inflamed tissues, blood and spleen, and their transfer is sufficient to induce disease without conventional T cells. Single-cell transcriptional and phenotyping analyses of isolated Tregs reveal expansion of non-lymphoid tissue (tissue-resident) Tregs expressing Th17-related genes, Helios, tissue-resident markers including CD103 and CD69, and a prominent NF-κB transcriptome. Thus, IKK2 regulates tissue-resident Treg differentiation, and overactivity drives dose-dependent skin and systemic inflammation.

Signaling via a CD27-TRAF2-SHP-1 axis during naive T cell activation promotes memory-associated gene regulatory networks.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

Systemically administered low-affinity HER2 CAR T cells mediate antitumor efficacy without toxicity.

BACKGROUND: The paucity of tumor-specific targets for chimeric antigen receptor (CAR) T-cell therapy of solid tumors necessitates careful preclinical evaluation of the therapeutic window for candidate antigens. Human epidermal growth factor receptor 2 (HER2) is an attractive candidate for CAR T-cell therapy in humans but has the potential for eliciting on-target off-tumor toxicity. We developed an immunocompetent tumor model of CAR T-cell therapy targeting murine HER2 (mHER2) and examined the effect of CAR affinity, T-cell dose, and lymphodepletion on safety and efficacy. METHODS: Antibodies specific for mHER2 were generated, screened for affinity and specificity, tested for immunohistochemical staining of HER2 on normal tissues, and used for HER2-targeted CAR design. CAR candidates were evaluated for T-cell surface expression and the ability to induce T-cell proliferation, cytokine production, and cytotoxicity when transduced T cells were co-cultured with mHER2+ tumor cells in vitro. Safety and efficacy of various HER2 CARs was evaluated in two tumor models and normal non-tumor-bearing mice. RESULTS: Mice express HER2 in the same epithelial tissues as humans, rendering these tissues vulnerable to recognition by systemically administered HER2 CAR T cells. CAR T cells designed with single-chain variable fragment (scFvs) that have high-affinity for HER2 infiltrated and caused toxicity to normal HER2-positive tissues but exhibited poor infiltration into tumors and antitumor activity. In contrast, CAR T cells designed with an scFv with low-affinity for HER2 infiltrated HER2-positive tumors and controlled tumor growth without toxicity. Toxicity mediated by high-affinity CAR T cells was independent of tumor burden and correlated with proliferation of CAR T cells post infusion. CONCLUSIONS: Our findings illustrate the disadvantage of high-affinity CARs for targets such as HER2 that are expressed on normal tissues. The use of low-affinity HER2 CARs can safely regress tumors identifying a potential path for therapy of solid tumors that exhibit high levels of HER2.

Atomic-Scale Elucidation of Unusually Distorted Dimeric Complexes Confined in a Zr-Based Metal-Organic Framework.

Nanoconfinement in metal-organic framework (MOF) pores can lead to the isolation of unusual or reactive metal complexes. However, MOFs that support the stabilization and precise structural elucidation of metal complexes and small metal clusters are rare. Here, we report a thermally and chemically stable zirconium-based MOF (University of Adelaide Material-1001, UAM-1001) with a high density of free bis-pyrazolyl units that can confine mono- and dinuclear metal complexes. The precursor MOF, UAM-1000, has a high degree of structural flexibility, but post synthetic modification with a bracing linker, biphenyl-4,4'-dicarboxylic acid, partially rigidifies the MOF (UAM-1001). This allows "matrix isolation" and detailed structural elucidation of postsynthetically added dimeric complexes bound within a tetradentate binding site formed by two linkers. Dimeric species [Co2Cl4], [Cu2Cl4], [Ni2Cl3(H2O)2]Cl, and [Rh2(CO)3Cl2] were successfully isolated in UAM-1001 and characterized by single-crystal X-ray diffraction. Comparison of the UAM-1001 isolated species with similar complexes in the solid state reveals that UAM-1001 can significantly distort the structures and enforce notably shorter metal-metal distances. For example, MOF tethering allows isolation of a [Cu2Cl4] complex that rapidly reacts with water in the solid state. The stability, porosity, and modulated flexibility of UAM-1001 provide an ideal platform material for the isolation and study of new dimeric complexes and their reactivity.

Versatile Tissue-Injectable Hydrogels with Extended Hydrolytic Release of Bioactive Protein Therapeutics.

Hydrogels generally have broad utilization in healthcare due to their tunable structures, high water content, and inherent biocompatibility. FDA-approved applications of hydrogels include spinal cord regeneration, skin fillers, and local therapeutic delivery. Drawbacks exist in the clinical hydrogel space, largely pertaining to inconsistent therapeutic exposure, short-lived release windows, and difficulties inserting the polymer into tissue. In this study, we engineered injectable, biocompatible hydrogels that function as a local protein therapeutic depot with a high degree of user-customizability. We showcase a PEG-based hydrogel functionalized with bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) handles for its polymerization and functionalization with a variety of payloads. Small-molecule and protein cargos, including chemokines and antibodies, were site-specifically modified with hydrolysable "azidoesters" of varying hydrophobicity via direct chemical conjugation or sortase-mediated transpeptidation. These hydrolysable esters afforded extended release of payloads linked to our hydrogels beyond diffusion; with timescales spanning days to months dependent on ester hydrophobicity. Injected hydrogels polymerize in situ and remain in tissue over extended periods of time. Hydrogel-delivered protein payloads elicit biological activity after being modified with SPAAC-compatible linkers, as demonstrated by the successful recruitment of murine T-cells to a mouse melanoma model by hydrolytically released murine CXCL10. These results highlight a highly versatile, customizable hydrogel-based delivery system for local delivery of protein therapeutics with payload release profiles appropriate for a variety of clinical needs.

Identifying cancer-associated leukocyte profiles using high-resolution flow cytometry screening and machine learning.

Background: Machine learning (ML) is a valuable tool with the potential to aid clinical decision making. Adoption of ML to this end requires data that reliably correlates with the clinical outcome of interest; the advantage of ML is that it can model these correlations from complex multiparameter data sets that can be difficult to interpret conventionally. While currently available clinical data can be used in ML for this purpose, there exists the potential to discover new "biomarkers" that will enhance the effectiveness of ML in clinical decision making. Since the interaction of the immune system and cancer is a hallmark of tumor establishment and progression, one potential area for cancer biomarker discovery is through the investigation of cancer-related immune cell signatures. Hence, we hypothesize that blood immune cell signatures can act as a biomarker for cancer progression. Methods: To probe this, we have developed and tested a multiparameter cell-surface marker screening pipeline, using flow cytometry to obtain high-resolution systemic leukocyte population profiles that correlate with detection and characterization of several cancers in murine syngeneic tumor models. Results: We discovered a signature of several blood leukocyte subsets, the most notable of which were monocyte subsets, that could be used to train CATboost ML models to predict the presence and type of cancer present in the animals. Conclusions: Our findings highlight the potential utility of a screening approach to identify robust leukocyte biomarkers for cancer detection and characterization. This pipeline can easily be adapted to screen for cancer specific leukocyte markers from the blood of cancer patient.

High-pressure single-crystal diffraction at the Australian Synchrotron.

A new high-pressure single-crystal diffraction setup has been designed and implemented at the Australian Synchrotron for collecting molecular and protein crystal structures. The setup incorporates a modified micro-Merrill-Bassett cell and holder designed specifically to fit onto the horizontal air-bearing goniometer, allowing high-pressure diffraction measurements to be collected with little to no modification of the beamline setup compared with ambient data collections. Compression data for the amino acid, L-threonine, and the protein, hen egg-white lysozyme, were collected, showcasing the capabilities of the setup.

Clostridium perfringens virulence factors are nonredundant activators of the NLRP3 inflammasome.

Inflammasome signaling is a central pillar of innate immunity triggering inflammation and cell death in response to microbes and danger signals. Here, we show that two virulence factors from the human bacterial pathogen Clostridium perfringens are nonredundant activators of the NLRP3 inflammasome in mice and humans. C. perfringens lecithinase (also known as phospolipase C) and C. perfringens perfringolysin O induce distinct mechanisms of activation. Lecithinase enters LAMP1+ vesicular structures and induces lysosomal membrane destabilization. Furthermore, lecithinase induces the release of the inflammasome-dependent cytokines IL-1ß and IL-18, and the induction of cell death independently of the pore-forming proteins gasdermin D, MLKL and the cell death effector protein ninjurin-1 or NINJ1. We also show that lecithinase triggers inflammation via the NLRP3 inflammasome in vivo and that pharmacological blockade of NLRP3 using MCC950 partially prevents lecithinase-induced lethality. Together, these findings reveal that lecithinase activates an alternative pathway to induce inflammation during C. perfringens infection and that this mode of action can be similarly exploited for sensing by a single inflammasome.

CX-ASAP: a high-throughput tool for the serial refinement and analysis of crystallographic data collected under varying conditions.

CX-ASAP is a new open-source software project designed to greatly reduce the time required to analyse crystallographic data collected under varying conditions. Scripted in Python3, CX-ASAP can automatically refine, finalize and analyse data collections with wide-ranging temperatures, pressures etc. This is achieved using a reference structure, allowing for quick identification of problems, phase changes and even model comparison. The modular design means that new features and customized scripts can be easily added, tailoring the capabilities to the specific needs of the user. It is envisioned that CX-ASAP will help to close the growing gap between fast collection times and slow data finalization.

Safety assessment scheme for menstrual cups and application for the evaluation of a menstrual cup comprised of medical grade silicone.

BACKGROUND: Ensuring menstrual cup safety is paramount, yet a menstrual cup safety assessment scheme is lacking. This paper presents a quadripartite scheme, showing how it can be applied. METHODS: The Tampax Menstrual Cup was evaluated in the safety assessment scheme: (1) Biocompatibility and chemical safety of cup constituents. Extractables were obtained under different use condition; exposure-based risk assessments (EBRA) were conducted for extractables exceeding thresholds of toxicological concern. (2) Physical impact to vaginal mucosa. After physical evaluations, the Tampax Cup and another cup were assessed in a randomised double-blinded, two-product, two-period cross-over clinical trial (65 women, mean age 34.2 years). (3) Impact to vaginal microbiota (in vitro mixed microflora assay and evaluation of vaginal swabs). (4) In vitro growth of Staphylococcus aureus and toxic shock syndrome toxin-1 (TSST-1) production. FINDINGS: Biocompatibility assessments and EBRA of cup constituents showed no safety concerns. In the randomised clinical trial, all potentially product-related adverse effects were mild, vaginal exams were unremarkable, no clinically relevant pH changes occurred, post-void residual urine volume with and without cup were similar, and self-reported measures of comfort along with reports of burning, itching and stinging between cups were comparable. Cup use had no effect on microbial growth in vitro or in the 62 subjects who completed the trial or on in vitro TSST-1 production. INTERPRETATION: The quadripartite safety assessment scheme allows evaluation of menstrual cup safety. The Tampax Cup is safe and well-tolerated upon intended use. As with all feminine hygiene products, post-market safety surveillance confirmed this conclusion. FUNDING: By Procter & Gamble.

Visible-Light-Responsive Self-Assembled Complexes: Improved Photoswitching Properties by Metal Ion Coordination.

A photoswitchable ligand based on azobenzene is self-assembled with palladium(II) ions to form a [Pd2 (E-L)4 ]4+ cage. Irradiation with 470 nm light results in the near-quantitative switching to a monomeric species [Pd(Z-L)2 ]2+ , which can be reversed by irradiation with 405 nm light, or heat. The photoswitching selectivity towards the metastable isomer is significantly improved upon self-assembly, and the thermal half-life is extended from 40 days to 850 days, a promising approach for tuning photoswitching properties.

Guest-Induced Multistep to Single-Step Spin-Crossover Switching in a 2-D Hofmann-Like Framework with an Amide-Appended Ligand.

A detailed study of the two-dimensional (2-D) Hofmann-like framework [Fe(furpy)2Pd(CN)4]·nG (furpy: N-(pyridin-4-yl)furan-2-carboxamide, G = H2O,EtOH (A·H2O,Et), and H2O (A·H2O)) is presented, including the structural and spin-crossover (SCO) implications of subtle guest modification. This 2-D framework is characterized by undulating Hofmann layers and an array of interlayer spacing environments─this is a strategic approach that we achieve by the inclusion of a ligand with multiple host-host and host-guest interaction sites. Variable-temperature magnetic susceptibility studies reveal an asymmetric multistep SCO for A·H2O,Et and an abrupt single-step SCO for A·H2O with an upshift in transition temperature of ∼75 K. Single-crystal analyses show a primitive orthorhombic symmetry for A·H2O,Et characterized by a unique FeII center─the multistep SCO character is attributed to local ligand orientation. Counterintuitively, A·H2O shows a triclinic symmetry with two inequivalent FeII centers that undergo a cooperative single-step high-spin (HS)-to-low-spin (LS) transition. We conduct detailed structure-function analyses to understand how the guest ethanol influences the delicate balance between framework communication and, therefore, the local structure and spin-state transition mechanism.

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration.

The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the gene's numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.

Immunization with a self-assembling nanoparticle vaccine displaying EBV gH/gL protects humanized mice against lethal viral challenge.

Epstein-Barr virus (EBV) is a cancer-associated pathogen responsible for 165,000 deaths annually. EBV is also the etiological agent of infectious mononucleosis and is linked to multiple sclerosis and rheumatoid arthritis. Thus, an EBV vaccine would have a significant global health impact. EBV is orally transmitted and has tropism for epithelial and B cells. Therefore, a vaccine would need to prevent infection of both in the oral cavity. Passive transfer of monoclonal antibodies against the gH/gL glycoprotein complex prevent experimental EBV infection in humanized mice and rhesus macaques, suggesting that gH/gL is an attractive vaccine candidate. Here, we evaluate the immunogenicity of several gH/gL nanoparticle vaccines. All display superior immunogenicity relative to monomeric gH/gL. A nanoparticle displaying 60 copies of gH/gL elicits antibodies that protect against lethal EBV challenge in humanized mice, whereas antibodies elicited by monomeric gH/gL do not. These data motivate further development of gH/gL nanoparticle vaccines for EBV.

High-sensitivity pattern discovery in large, paired multiomic datasets.

MOTIVATION: Modern biological screens yield enormous numbers of measurements, and identifying and interpreting statistically significant associations among features are essential. In experiments featuring multiple high-dimensional datasets collected from the same set of samples, it is useful to identify groups of associated features between the datasets in a way that provides high statistical power and false discovery rate (FDR) control. RESULTS: Here, we present a novel hierarchical framework, HAllA (Hierarchical All-against-All association testing), for structured association discovery between paired high-dimensional datasets. HAllA efficiently integrates hierarchical hypothesis testing with FDR correction to reveal significant linear and non-linear block-wise relationships among continuous and/or categorical data. We optimized and evaluated HAllA using heterogeneous synthetic datasets of known association structure, where HAllA outperformed all-against-all and other block-testing approaches across a range of common similarity measures. We then applied HAllA to a series of real-world multiomics datasets, revealing new associations between gene expression and host immune activity, the microbiome and host transcriptome, metabolomic profiling and human health phenotypes. AVAILABILITY AND IMPLEMENTATION: An open-source implementation of HAllA is freely available at http://huttenhower.sph.harvard.edu/halla along with documentation, demo datasets and a user group. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Regulation of Multistep Spin Crossover Across Multiple Stimuli in a 2-D Framework Material.

We investigate the effects of a broad array of external stimuli on the structural, spin-crossover (SCO) properties and nature of the elastic interaction within the two-dimensional Hofmann framework material [Fe(cintrz)2Pd(CN)4]·guest (cintrz = N-cinnamalidene 4-amino-1,2,4-triazole; A·guest; guest = 3H2O, 2H2O, and Ø). This framework exhibits a delicate balance between ferro- and antiferro-elastic interaction characters; we show that manipulation of the pore contents across guests = 3H2O, 2H2O, and Ø can be exploited to regulate this balance. In A·3H2O, the dominant antiferroelastic interaction character between neighboring FeII sites sees the low-temperature persistence of the mixed spin-state species {HS-LS} for {Fe1-Fe2} (HS = high spin, LS = low spin). Elastic interaction strain is responsible for stabilizing the {HS-LS} state and can be overcome by three mechanisms: (1) partial (2H2O) or complete (Ø) guest removal, (2) irradiation via the reverse light-induced excited spin-state trapping (LIESST) effect (λ = 830 nm), and (3) the application of external hydrostatic pressure. Combining experimental data with elastic models presents a clear interpretation that while guest molecules cause a negative chemical pressure, they also have consequences for the elastic interactions between metals beyond the simple chemical pressure picture typically proposed.

The Sinorhizobium meliloti Nitrogen Stress Response Changes Radically in the Face of Concurrent Phosphate Stress.

Expression of hundreds of S. meliloti genes changed more than two-fold in response to either nitrogen or phosphate limitation. When these two stresses were applied together, stress responsive gene expression shifted dramatically. In particular, the nitrogen stress response in the presence of phosphate stress had only 30 of about 350 genes in common with the 280 genes that responded to nitrogen stress with adequate phosphate. Expression of sRNAs was also altered in response to these stresses. 82% of genes that responded to nitrogen stress also responded to phosphate stress, including 20 sRNAs. A subset of these sRNAs is known to be chaperoned by the RNA binding protein, Hfq. Hfq had previously been shown to influence about a third of the genes that responded to both nitrogen and phosphate stresses. Phosphate limitation influenced changes in gene expression more than nitrogen limitation and, when both stresses were present, phosphate stress sometimes reversed the direction of some of the changes induced by nitrogen stress. These nutrient stress responses are therefore context dependent.

Machine learning predicts cancer subtypes and progression from blood immune signatures.

Clinical adoption of immune checkpoint inhibitors in cancer management has highlighted the interconnection between carcinogenesis and the immune system. Immune cells are integral to the tumour microenvironment and can influence the outcome of therapies. Better understanding of an individual's immune landscape may play an important role in treatment personalisation. Peripheral blood is a readily accessible source of information to study an individual's immune landscape compared to more complex and invasive tumour bioipsies, and may hold immense diagnostic and prognostic potential. Identifying the critical components of these immune signatures in peripheral blood presents an attractive alternative to tumour biopsy-based immune phenotyping strategies. We used two syngeneic solid tumour models, a 4T1 breast cancer model and a CT26 colorectal cancer model, in a longitudinal study of the peripheral blood immune landscape. Our strategy combined two highly accessible approaches, blood leukocyte immune phenotyping and plasma soluble immune factor characterisation, to identify distinguishing immune signatures of the CT26 and 4T1 tumour models using machine learning. Myeloid cells, specifically neutrophils and PD-L1-expressing myeloid cells, were found to correlate with tumour size in both the models. Elevated levels of G-CSF, IL-6 and CXCL13, and B cell counts were associated with 4T1 growth, whereas CCL17, CXCL10, total myeloid cells, CCL2, IL-10, CXCL1, and Ly6Cintermediate monocytes were associated with CT26 tumour development. Peripheral blood appears to be an accessible means to interrogate tumour-dependent changes to the host immune landscape, and to identify blood immune phenotypes for future treatment stratification.