Targeted desialylation and cytolysis of tumour cells by fusing a sialidase to a bispecific T-cell engager.

Bispecific T-cell engagers (BiTEs) bring together tumour cells and cytotoxic T cells by binding to specific cell-surface tumour antigens and T-cell receptors, and have been clinically successful for the treatment of B-cell malignancies. Here we show that a BiTE-sialidase fusion protein enhances the susceptibility of solid tumours to BiTE-mediated cytolysis of tumour cells via targeted desialylation-that is, the removal of terminal sialic acid residues on glycans-at the BiTE-induced T-cell-tumour-cell interface. In xenograft and syngeneic mouse models of leukaemia and of melanoma and breast cancer, and compared with the parental BiTE molecules, targeted desialylation via the BiTE-sialidase fusion proteins enhanced the formation of immunological synapses, T-cell activation and T-cell-mediated tumour-cell cytolysis in the presence of the target antigen. The targeted desialylation of tumour cells may enhance the potency of therapies relying on T-cell engagers.

Control of the antitumour activity and specificity of CAR T cells via organic adapters covalently tethering the CAR to tumour cells.

On-target off-tumour toxicity limits the anticancer applicability of chimaeric antigen receptor (CAR) T cells. Here we show that the tumour-targeting specificity and activity of T cells with a CAR consisting of an antibody with a lysine residue that catalytically forms a reversible covalent bond with a 1,3-diketone hapten can be regulated by the concentration of a small-molecule adapter. This adapter selectively binds to the hapten and to a chosen tumour antigen via a small-molecule binder identified via a DNA-encoded library. The adapter therefore controls the formation of a covalent bond between the catalytic antibody and the hapten, as well as the tethering of the CAR T cells to the tumour cells, and hence the cytotoxicity and specificity of the cytotoxic T cells, as we show in vitro and in mice with prostate cancer xenografts. Such small-molecule switches of T-cell cytotoxicity and specificity via an antigen-independent 'universal' CAR may enhance the control and safety profile of CAR-based cellular immunotherapies.

Measuring Youth Perceptions of Being Known and Loved and Positive Youth Development: Cross-National Findings from Rwanda and El Salvador.

Background: Dynamic, relational developmental systems-based models of development emphasize that developmentally-nurturant youth-adult relationships elicit in youth perceptions of being known and loved. Although such perceptions are foundations of positive youth development (PYD), such measures do not exist. Objective: We sought to create a theoretically-predicated measure of youth perceptions of being known and loved by capitalizing on data sets in two countries (Rwanda and El Salvador) wherein a multi-national study of PYD was being conducted by Compassion International (CI). Method: With Rwanda data (n = 1,204, M age = 11.84, 50% CI-supported), exploratory and confirmatory factor analyses enabled refining the measure for robustness and parsimony. Measures of intentional self-regulation, hopeful future expectations, transcendence, and contribution were used for validation of the known and loved measure within the nomological net of constructs proposed in the Lerner and Lerner PYD model. Confirmatory factor analyses supported the use of the model within the El Salvador data set (n = 1,205, M age = 13.03, 51% CI-supported). Results: Robust psychometric properties were established in both national settings. Measurement invariance was found across age, gender, urban-rural location, CI-enrollment status, and nations, and involved both mean differences and correlations among latent factors. Conclusions: The results provide evidence for a theory-predicated measure of youth perceptions of being known and loved and that scores for this construct covary within a nomological net specified in the Lerner and Lerner model of PYD. These findings serve international development organizations seeking theory-predicated measures for use in evaluating PYD programs in low- and middle-income countries. Supplementary Information: The online version contains supplementary material available at 10.1007/s10566-022-09725-6.

Switchable targeting of solid tumors by BsCAR T cells.

The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2+ ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.

Does Prior Night's Sleep Impact Next Day's Executive Functioning? It Depends on an Individual's Average Sleep Quality.

Executive functioning (EF) is a series of fundamental goal-directed cognitive abilities that enable effective learning. Differences in daily sleep quality may covary with fluctuations in EF among youth. Most studies linking sleep to EF rely on between-person differences and average effects for the sample. This study employed an intensive longitudinal design and examined the within-person relations between self-reported prior night's sleep quality and next day's EF. Students from Grades 4 to 12 (M age= 14.60, SD = 2.53) completed three behavioral EF tasks repeatedly across approximately one semester. The final analytic sample included 2898 observations embedded in 73 participants. Although, on average, sleep did not significantly covary with EF, there was heterogeneity in within-person sleep-EF relations. Moreover, individuals' average sleep quality moderated within-person effects. For individuals with low mean sleep quality, a better-than-usual sleep quality was linked to better EF performance. However, for individuals with high mean sleep quality, better-than-usual sleep quality was linked to worse EF performance. Understanding person-specific relations between sleep and EF can help educators optimize EF and learning on a daily basis and produce positive academic outcomes across longer time periods.

Neutralizing Antibodies to SARS-CoV-2 Selected from a Human Antibody Library Constructed Decades Ago.

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, a combinatorial human antibody library constructed 20 years before the coronavirus disease 2019 (COVID-19) pandemic is used to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these three antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in their crystal structures with SARS-CoV-2 spike receptor binding domain. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of these antibodies with respect to their reactivity with SARS-CoV-2.

Positive Youth Development in 2020: Theory, Research, Programs, and the Promotion of Social Justice.

We use Hamilton's (1999) tripartite conception of the positive youth development (PYD) literature - that is, PYD as a theoretical construct, PYD as a frame for program design, and PYD as an instance of specific youth development programs - as a framework for reviewing scholarship involved in the PYD field across the second decade of the 21st century. Advances were made in all three domains and, as well, new issues emerged; chief among them was a focus on the promotion of social justice. We discuss ways in which social justice issues are being addressed within each of these domains and we present a vision for enhancing the PYD-social justice relation in future scholarship involving theory, research, program design, and community-based PYD programs.

Antigen-Specific Stimulation and Expansion of CAR-T Cells Using Membrane Vesicles as Target Cell Surrogates.

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low-efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. To develop a specific and improved protocol for CAR-T cell expansion, cell-derived membrane vesicles are taken advantage of, and the simple structural demands of the CAR-antigen interaction. This novel approach is to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens, and then use them for stimulation and expansion of CAR-T cells. The data presented in this article clearly demonstrate that this protocol produced antigen-specific vesicles with the capacity to induce stronger stimulation, proliferation, and functional activity of CAR-T cells than is possible with existing protocols. It is predicted that this new methodology will significantly advance the ability to obtain improved populations of functional CAR-T cells for therapy.

A new immunochemical strategy for triple-negative breast cancer therapy.

Triple-negative breast cancer (TNBC) is a highly diverse group of malignant neoplasms which tend to have poor outcomes, and the development of new targets and strategies to treat these cancers is sorely needed. Antibody-drug conjugate (ADC) therapy has been shown to be a promising targeted therapy for treating many cancers, but has only rarely been tried in patients with TNBC. A major reason the efficacy of ADC therapy in the setting of TNBC has not been more fully investigated is the lack of appropriate target molecules. In this work we were able to identify an effective TNBC target for use in immunotherapy. We were guided by our previous observation that in some breast cancer patients the protein tropomyosin receptor kinase B cell surface protein (TrkB) had become immunogenic, suggesting that it was somehow sufficiently chemically different enough (presumably by mutation) to escaped immune tolerance. We postulated that this difference might well offer a means for selective targeting by antibodies. We engineered site-specific ADCs using a dual variable domain (DVD) format which combines anti-TrkB antibody with the h38C2 catalytic antibody. This format enables rapid, one-step, and homogeneous conjugation of ß-lactam-derivatized drugs. Following conjugation to ß-lactam-derivatized monomethyl auristatin F, the TrkB-targeting DVD-ADCs showed potency against multiple breast cancer cell lines, including TNBC cell lines. In addition, our isolation of antibody that specifically recognized the breast cancer-associated mutant form of TrkB, but not the wild type TrkB, indicates the possibility of further refining the selectivity of anti-TrkB DVD-ADCs, which should enhance their therapeutic index. These results confirmed our supposition that TrkB is a potential target for immunotherapy for TNBC, as well as for other cancers with mutated cell surface proteins.

Selection of a picomolar antibody that targets CXCR2-mediated neutrophil activation and alleviates EAE symptoms.

Receptors and their ligands are important therapeutic targets for about one third of marketed drugs. Here, we describe an epitope-guided approach for selection of antibodies that modulate cellular signaling of targeted receptors. We chose CXC chemokine receptor 2 (CXCR2) in the G-protein coupled receptor superfamily as receptor and a CXCR2 N-terminal peptide for antibody selection. We obtain a highly selective, tight-binding antibody from a 1011-member antibody library using combinatorial enrichment. Structural and Hydrogen-Deuterium-Exchange mass spectrometry analyses demonstrate antibody interaction with an N-terminal region of CXCR2 that is part of the IL-8 epitope. The antibody strongly inhibits IL-8-induced and CXCR2-mediated neutrophil chemotaxis in vitro and alleviates hCXCR2-dependent experimental autoimmune encephalomyelitis symptoms in mice. As inappropriate neutrophil migration accompanies many diseases including inflammatory bowel disease, glomerulonephritis, allergic asthma, chronic obstructive pulmonary disease, and cancer, this antibody has potential for development as a therapeutic agent, akin to anti-TNF antibodies. However, an important difference here is that the antibody targets the chemokine receptor and competes with natural ligand, rather than targeting the ligand itself.

Antibody Libraries as Tools to Discover Functional Antibodies and Receptor Pleiotropism.

Most antibodies currently in use have been selected based on their binding affinity. However, nowadays, antibodies that can not only bind but can also alter the function of cell surface signaling components are increasingly sought after as therapeutic drugs. Therefore, the identification of such functional antibodies from a large antibody library is the subject of intensive research. New methods applied to combinatorial antibody libraries now allow the isolation of functional antibodies in the cellular environment. These selected agonist antibodies have provided new insights into important issues of signal transduction. Notably, when certain antibodies bind to a given receptor, the cell fate induced by them may be the same or different from that induced by natural agonists. In addition, combined with phenotypic screening, this platform allows us to discover unexpected experimental results and explore various phenomena in cell biology, such as those associated with stem cells and cancer cells.

Stereo- and regiodefined DNA-encoded chemical libraries enable efficient tumour-targeting applications.

The encoding of chemical compounds with amplifiable DNA tags facilitates the discovery of small-molecule ligands for proteins. To investigate the impact of stereo- and regiochemistry on ligand discovery, we synthesized a DNA-encoded library of 670,752 derivatives based on 2-azido-3-iodophenylpropionic acids. The library was selected against multiple proteins and yielded specific ligands. The selection fingerprints obtained for a set of protein targets of pharmaceutical relevance clearly showed the preferential enrichment of ortho-, meta- or para-regioisomers, which was experimentally verified by affinity measurements in the absence of DNA. The discovered ligands included novel selective enzyme inhibitors and binders to tumour-associated antigens, which enabled conditional chimeric antigen receptor T-cell activation and tumour targeting.

DNA-Encoded Libraries: Hydrazide as a Pluripotent Precursor for On-DNA Synthesis of Various Azole Derivatives.

DNA-encoded combinatorial chemical library (DEL) technology, an approach that combines the power of genetics and chemistry, has emerged as an invaluable tool in drug discovery. Skeletal diversity plays a fundamental importance in DEL applications, and relies heavily on novel DNA-compatible chemical reactions. We report herein a phylogenic chemical transformation strategy using DNA-conjugated benzoyl hydrazine as a common versatile precursor in azole chemical expansion of DELs. DNA-compatible reactions deriving from the common benzoyl hydrazine precursor showed excellent functional group tolerance with exceptional efficiency in the synthesis of various azoles, including oxadiazoles, thiadiazoles, and triazoles, under mild reaction conditions. The phylogenic chemical transformation strategy provides DELs a facile way to expand into various unique chemical spaces with privileged scaffolds and pharmacophores.

Avidity-Based Selection of Tissue-Specific CAR-T Cells from a Combinatorial Cellular Library of CARs.

Using T-cell chimeric antigen receptors (CAR-T) to activate and redirect T cells to tumors expressing the cognate antigen represents a powerful approach in cancer therapy. However, normal tissues with low expression of tumor-associated antigens (TAAs) can be mistargeted, resulting in severe side effects. An approach using a collection of T cells expressing a diverse, 106-member combinatorial cellular library of CARs, in which members can be specifically enriched based on avidity for cell membrane antigens, is reported. Using CD38 as the target antigen, an efficient and effective selection of CARs specifically recognizing CD38+ tumor cells is demonstrated. These selected CAR-T's produce cytokines known to be associated with T cell activation in a CD38 expression-dependent manner. This avidity-based selection endows the engineered T cells with minimal off-tumor effects, while retaining robust antitumor efficacy both in vitro and in vivo. The described method may facilitate the application of CAR-T therapy to TAAs previously considered undruggable.

Intraindividual Fluctuations in Sleep Predict Subsequent Goal Setting in Adolescents.

The purpose of this study was to investigate within- and between-person associations between sleep and subsequent goal setting in adolescents. We conducted an intensive repeated measures longitudinal study to assess intra- and inter-individual associations between sleep and goal setting and potential moderators of such associations. Thirty-nine seventh through 12th graders reported on their sleep quality and propensity to set goals in their daily lives several times per week for approximately four months. We used a combination of multilevel modeling with time-varying covariates and centering techniques to partition within- and between-person variance. We found significant and positive associations between sleep and goal setting within individuals, but no such associations between individuals. That is, students were more likely to set goals for their work after getting a good night's sleep relative to their own average sleep quality, but getting good sleep on average relative to other individuals showed no association with average goal setting. These relationships were not moderated by participant age, gender, or sociodemographic status as indexed by maternal education. Differences in average sleep between adolescents matters less for their propensity to set goals than whether they experienced better- or worse-than-usual sleep the previous night given their own average. This finding represents the first evidence documenting effects of sleep on goal setting, which is an important psychological precursor to many youth behavioral and achievement outcomes. Our findings highlight the individuality of sleep needs and point to new directions for sleep-related practice and policy aimed at youth.

Tough Teams and Optimistic Individuals: The Intersecting Roles of Group and Individual Attributes in Helping to Predict Physical Performance.

This study tested the effects of individual and group-level characteristics on performance during a mandatory and challenging physical education course at the United States Military Academy (USMA). We focused on attributes related to mental toughness, and examined both self-report and utilized an other-rating scale that measures mental toughness-related characteristics and is important to USMA generally. We examined course scores for 5,581 first-year students over five academic years, accounted for background physical fitness, and determined how mental toughness attributes at the group and individual-level contributed to overall course score and scores on constituent events (e.g. obstacle course, rope climbing). Self-reported optimism, self-reported resilience, and mental toughness items from a peer rating scale, but not self-reported grit, significantly improved course performance. The average score across class section on optimism or the peer rating scale also positively covaried with course score, over and above the individual-level impact of that attribute. Analyses of individual events demonstrated that "group-level character" was important for some events, whereas individual attributes were most important for others. Findings suggested an emergent group character capable of influencing individual physical performance scores. Being a member of a tough group may have comparable effects to individual mental toughness.

gem-Difluoromethylene Alkyne-Enabled Diverse C-H Functionalization and Application to the on-DNA Synthesis of Difluorinated Isocoumarins.

Using gem-difluoromethylene alkynes as effectors, unprecedented diverse C-H activation/[4+2] annulations of simple benzoic acids are reported. The chemodivergent reaction outcomes are well-tuned by Rh/Ir-catalyzed system; in the RhIII catalysis, 3-alkenyl-1H-isochromen-1-one and 3,4-dialkylideneisochroman-1-one skeletons are afforded in a solvent-dependent manner whereas difluoromethylene-substituted 1H-isochromen-1-ones are generated under the IrIII -catalyzed system. Mechanistic studies revealed that unusually double ß-F eliminations and fluorine effect-induced regioselective reductive elimination are independently involved to enable distinct reaction modes for divergent product formations. Besides, synthetic application in both the derivatization of obtained diene products and the on-DNA synthesis of DNA-tagged difluorinated isocoumarin have been demonstrated, which manifested great potential for synthetic utility of the developed protocols.

Multiscale computation delivers organophosphorus reactivity and stereoselectivity to immunoglobulin scavengers.

Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors.