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Cancer immunotherapy has flourished over the last 10–15 years, transforming the practice of oncology and providing long-term clinical benefit to some patients. During this time, three distinct classes of immune checkpoint inhibitors, chimeric antigen receptor-T cell therapies specific for two targets, and two distinct classes of bispecific T cell engagers, a vaccine, and an oncolytic virus have joined cytokines as a standard of cancer care. At the same time, scientific progress has delivered vast amounts of new knowledge. For example, advances in technologies such as single-cell sequencing and spatial transcriptomics have provided deep insights into the immunobiology of the tumor microenvironment. With this rapid clinical and scientific progress, the field of cancer immunotherapy is currently at a critical inflection point, with potential for exponential growth over the next decade. Recognizing this, the Society for Immunotherapy of Cancer convened a diverse group of experts in cancer immunotherapy representing academia, the pharmaceutical and biotechnology industries, patient advocacy, and the regulatory community to identify current opportunities and challenges with the goal of prioritizing areas with the highest potential for clinical impact. The consensus group identified seven high-priority areas of current opportunity for the field: mechanisms of antitumor activity and toxicity; mechanisms of drug resistance; biomarkers and biospecimens; unique aspects of novel therapeutics; host and environmental interactions; premalignant immunity, immune interception, and immunoprevention; and clinical trial design, endpoints, and conduct. Additionally, potential roadblocks to progress were discussed, and several topics were identified as cross-cutting tools for optimization, each with potential to impact multiple scientific priority areas. These cross-cutting tools include preclinical models, data curation and sharing, biopsies and biospecimens, diversification of funding sources, definitions and standards, and patient engagement. Finally, three key guiding principles were identified that will both optimize and maximize progress in the field. These include engaging the patient community; cultivating diversity, equity, inclusion, and accessibility; and leveraging the power of artificial intelligence to accelerate progress. Here, we present the outcomes of these discussions as a strategic vision to galvanize the field for the next decade of exponential progress in cancer immunotherapy.

Objective This study evaluated the relationship between physical literacy (PL) and muscular fitness (MF) as well as muscle-strengthening activities (MSA) in adolescents.Methods A secondary cross-sectional study included 823 adolescents (45.1% boys) from the Eating Healthy and Daily Life Activities Study. The Spanish Perceived Physical Literacy Instrument for Adolescents (S-PPLI) assessed the participants’ perceived PL. The evaluation of MF in the young population was conducted using the Assessing the Levels of Physical Activity and Fitness (ALPHA-FIT) test battery. MSA were assessed by the following question: ‘In the past week, how many days did you exercise to strengthen or tone the muscle, such as through push-ups, sit-ups or lifting weights?’ To examine the associations between S-PPLI scores and handgrip strength, standing long jump and MSA days in adolescents, we employed generalised additive models.Results Participants with high perceived physical literacty (PPL) had the highest handgrip strength (mean=25.1; PPL had the highest handgrip strength (mean=25.1; bias-corrected and accelerated (BCa) bootstrapped 95% CI 24.2 to 26.0) and standing long jump (mean=149.7; confidence interval (CI) 24.2 to 26.0) and standing long jump (mean=149.7; 24.2 to 26.0) and standing long jump (mean=149.7; BCa bootstrapped 95% CI 145.8 to 153.5). Conversely, those adolescents with low PPL had the lowest handgrip strength (mean=23.1; BCa bootstrapped 95% CI 22.3 to 23.8) and standing long jump (mean=137.3; BCa bootstrapped 95% CI 133.9 to 140.6). For MSA, adolescents with high PPL had the highest mean of MSA days (mean=3.0; BCa bootstrapped 95% CI 2.8 to 3.3), while the lowest mean was observed for those with low PPL (mean=2.0; BCa bootstrapped 95% CI 1.8 to 2.2).Conclusion A high level of PL was associated with higher levels of MF and MSA among Spanish adolescents.

The myeloma cell surface proteome regulates plasma cell biology and delineates therapy targets. Here, the authors profile the myeloma surfaceome at baseline and in drug resistance, finding the potential target CCR10, and include a streamlined approach to primary sample analysis.

Abstract Epidural anesthesia requires injection of anesthetic into the epidural space in the spine. Accurate placement of the epidural needle is a major challenge. To address this, we developed a forward-view endoscopic optical coherence tomography (OCT) system for real-time imaging of the tissue in front of the needle tip during the puncture. We tested this OCT system in porcine backbones and developed a set of deep learning models to automatically process the imaging data for needle localization. A series of binary classification models were developed to recognize the five layers of the backbone, including fat, interspinous ligament, ligamentum flavum, epidural space, and spinal cord. The classification models provided an average classification accuracy of 96.65%. During puncture, it is important to maintain a safe distance between the needle tip and the dura mater. Regression models were developed to estimate that distance based on the OCT imaging data. Based on the Inception architecture, our models achieved a mean absolute percentage error of 3.05% ± 0.55%. Overall, our results validated the technical feasibility of using this novel imaging strategy to automatically recognize different tissue structures and measure the distances ahead of the needle tip during the epidural needle placement.

KRAS is commonly mutated at codon 12 in several cancer types, offering a unique opportunity for the development of neoantigen-targeted immunotherapy. Here the authors present a pipeline for the prediction, identification and validation of HLA class-I restricted mutant KRAS G12 peptides, leading to the generation of mutant KRAS-specific T cell receptors for adoptive T cell immunotherapy.

BackgroundImproved survival rates for children and adolescents diagnosed with cancer call for novel strategies for reducing short- and long-term treatment-related side effects. These include the physical and metabolic sequelae that are exacerbated by sedentary behavior and treatment-induced toxicities. We aim to investigate the effect of an integrative neuromuscular training intervention during the first 6 months of anti-cancer treatment primarily on muscle strength, and secondarily on exercise capacity, physical function, markers of metabolic syndrome, dysmetabolism, and health-related quality of life during and after ended treatment.MethodsOne hundred and twenty-seven children and adolescents, newly diagnosed with malignant and benign neoplasia, aged 6–17 years, and treated with chemotherapy or radiation will be randomized to either the intervention or the control arm of the study. The intervention group will, in addition to usual care, be offered a combination of 6 months of supervised physical exercise (integrative neuromuscular training) and home-based exercise. The active control group will, in addition to usual care, receive information along an unsupervised written home-based training program. All participants, including parents, will receive information about the importance of physical exercise during the course of cancer treatment, at the start of treatment, and in 5 monthly sessions. The primary outcome is measured in terms of isometric quadriceps muscle strength. Secondary outcomes include muscle strength and endurance, markers of metabolic syndrome and dysmetabolism, exercise capacity, physical function and activity, days of hospitalization, and health-related quality of life. Assessment will be conducted at treatment initiation (baseline), at 3 and 6 months after inclusion, and 1 month and 1 year after ended treatment. The primary endpoint for lower-body muscle strength is at 6 months after treatment initiation. The effects of the intervention will be evaluated through a constrained linear mixed model.DiscussionThis national randomized controlled study has the potential to provide new knowledge concerning the short- and long-term effects of a novel, inclusive approach for youth exercise programming (integrative neuromuscular exercise) in children and adolescents during anti-cancer treatment. Using a pragmatic, low-cost, and time-efficient training design, this intervention can be easily adapted to both hospital and home settings.Clinical Trial RegistrationClinicalTrials.gov (NCT04706676), first released January 5, 2021.

Abstract Background Depression affects approximately 7.1% of the United States population every year and has an annual economic burden of over $210 billion dollars. Several recent studies have sought to investigate the pathophysiology of depression utilizing focused cerebrospinal fluid (CSF) and serum analysis. Inflammation and metabolic dysfunction have emerged as potential etiological factors from these studies. A dysregulation in the levels of inflammatory proteins such as IL-12, TNF, IL-6 and IFN-γ have been found to be significantly correlated with depression. Methods CSF samples were obtained from 15 patients, seven with major depressive disorder and eight age- and gender-matched non-psychiatric controls. CSF protein profiles were obtained using quantitative mass spectrometry. The data were analyzed by Progenesis QI proteomics software to identify significantly dysregulated proteins. The results were subjected to bioinformatics analysis using the Ingenuity Pathway Analysis suite to obtain unbiased mechanistic insight into biologically relevant interactions and pathways. Results Several dysregulated proteins were identified. Bioinformatics analysis indicated that the potential disorder/disease pathways include inflammatory response, metabolic disease and organismal injury. Molecular and cellular functions that were affected include cellular compromise, cell-to-cell signaling & interaction, cellular movement, protein synthesis, and cellular development. The major canonical pathway that was upregulated was acute phase response signaling. Endogenous upstream regulators that may influence dysregulation of proinflammatory molecules associated with depression are interleukin-6 (IL-6), signal transducer and activator of transcription 3 (STAT3), oncostatin M, PR domain zinc finger protein 1 (PRDM1), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). Conclusions The proteome profiling data in this report identifies several potential biological functions that may be involved in the pathophysiology of major depressive disorder. Future research into how the differential expression of these proteins is involved in the etiology and severity of depression will be important.

T cells engineered with chimeric antigen receptors (CARs) have emerged as a potent new class of therapeutics for cancer, based on their remarkable potency in blood cancers. Since the first clinical reports of their efficacy emerged 7 years ago, investigators have focused on the mechanisms and properties that make CARs effective or toxic, and their effects on T cell biology. Novel CAR designs coupled with improvements in gene transfer technology, incorporating advances in gene editing, have the potential to increase access to engineered cell therapies, as well as improve their potency in solid tumors. Keywords: CAR T cells, immunotherapy, chimeric antigen receptors, cancer

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells’ efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells. Keywords: glioblastoma, chimeric antigen receptor, CAR, IL-13Rα2, minibody, canine, immune checkpoint blockade, PD-1, CTLA-4, TIM-3

ABSTRACT Bacterial biofilms are major contributors to chronic infections in humans. Because they are recalcitrant to conventional therapy, they present a particularly difficult treatment challenge. Identifying factors involved in biofilm development can help uncover novel targets and guide the development of antibiofilm strategies. Pseudomonas aeruginosa causes surgical site, burn wound, and hospital-acquired infections and is also associated with aggressive biofilm formation in the lungs of cystic fibrosis patients. A potent but poorly understood contributor to P. aeruginosa virulence is the ability to produce outer membrane vesicles (OMVs). OMV trafficking has been associated with cell-cell communication, virulence factor delivery, and transfer of antibiotic resistance genes. Because OMVs have almost exclusively been studied using planktonic cultures, little is known about their biogenesis and function in biofilms. Several groups have shown that Pseudomonas quinolone signal (PQS) induces OMV formation in P. aeruginosa. Our group described a biophysical mechanism for this and recently showed it is operative in biofilms. Here, we demonstrate that PQS-induced OMV production is highly dynamic during biofilm development. Interestingly, PQS and OMV synthesis are significantly elevated during dispersion compared to attachment and maturation stages. PQS biosynthetic and receptor mutant biofilms were significantly impaired in their ability to disperse, but this phenotype was rescued by genetic complementation or exogenous addition of PQS. Finally, we show that purified OMVs can actively degrade extracellular protein, lipid, and DNA. We therefore propose that enhanced production of PQS-induced OMVs during biofilm dispersion facilitates cell escape by coordinating the controlled degradation of biofilm matrix components. IMPORTANCE Treatments that manipulate biofilm dispersion hold the potential to convert chronic drug-tolerant biofilm infections from protected sessile communities into released populations that are orders-of-magnitude more susceptible to antimicrobial treatment. However, dispersed cells often exhibit increased acute virulence and dissemination phenotypes. A thorough understanding of the dispersion process is therefore critical before this promising strategy can be effectively employed. Pseudomonas quinolone signal (PQS) has been implicated in early biofilm development, but we hypothesized that its function as an outer membrane vesicle (OMV) inducer may contribute at multiple stages. Here, we demonstrate that PQS and OMVs are differentially produced during Pseudomonas aeruginosa biofilm development and provide evidence that effective biofilm dispersion is dependent on the production of PQS-induced OMVs, which likely act as delivery vehicles for matrix-degrading enzymes. These findings lay the groundwork for understanding OMV contributions to biofilm development and suggest a model to explain the controlled matrix degradation that accompanies biofilm dispersion in many species.

T-cell therapies have made significant improvements in cancer treatment over the last decade. One cellular therapy utilizing T-cells involves the use of a chimeric MHC-independent antigen-recognition receptor, typically referred to as a chimeric antigen receptor (CAR). CAR molecules, while mostly limited to the recognition of antigens on the surface of tumor cells, can also be utilized to exploit the diverse repertoire of macromolecules targetable by antibodies, which are incorporated into the CAR design. Leaning into this expansion of target macromolecules will enhance the diversity of antigens T-cells can target and may improve the tumor-specificity of CAR T-cell therapy. This review explores the types of macromolecules targetable by T-cells through endogenous and synthetic antigen-specific receptors.

The experience of being bullied in childhood or adolescence affects health into adulthood and is a public health crisis. Particularly affected are sexual minority young adults who are at the greatest risk for severe and violent bullying, HIV seroconversion, and onset of a substance use disorder. Although the scholarly work in the area of bullying victimization has made great gains over the past few years via improved sampling and methodological rigor, most of the focus of health research in this area has been on prevention efforts. The purpose of the current study was to inform the development of a transdiagnostic integrated treatment platform that will focus on mental and physical health outcomes that include sexual risk taking and substance abuse. This study involved conducting four focus groups with HIV-uninfected gay and bisexual men, aged 18 to 26 years, in order to examine treatment needs and preferences and further develop an evidence-based intervention. Four themes emerged from the analysis of transcripts: (a) learning about bullying and psychopathology, (b) coping with bullying, (c) experiencing psychopathology as a consequence of bullying, and (d) tailoring psychosocial interventions to address health sequelae linked with bullying. These themes provided a solid foundation to develop and test an intervention to address key health risks among men who have sex with men (MSM) with a history of being bullied and recent sexual risk taking and substance abuse.

This review aimed to synthesize the most up-to-date evidence regarding the prevalence of urinary incontinence (UI) among adolescent female athletes. We conducted a systematic review of studies regarding UI in female athletes less than 19 years of age. This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRIMSA). The electronic databases of PubMed, Embase, Cochrane Central Register of Controlled Trials (CENTRAL), Scopus, and Web of Science (WOS) were searched between October and November 2020. After blinded peer evaluation, a total of 215 studies were identified and nine were included. Risk of bias was assessed using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist. This review identified a prevalence of UI in adolescent female athletes between 18% to 80% with an average of 48.58%. The most prevalent sports were trampolining followed by rope skipping. The prevalence of UI among adolescent female athletes practicing impact sports was significantly prevalent. There is a need for further research, education, and targeted interventions for adolescent female athletes with UI.

Much of the evidence examining temporal trends in fitness among youth has found a decrease in measures of muscular strength and muscular power over recent decades. The aim of this study was to examine trends in lower body muscular power in Spanish boys over 47 years. In 1969 140 boys (10–11 years; body mass index = 19.24, SD = 2.91 kg/m2) and in 2016, 113 boys (10–11 years; body mass index = 19.20, SD = 3.15 kg/m2) were recruited. Lower body power was assessed using the vertical jump (VJ) and standing long jump (SLJ) tests. Significant differences and a large effect size were shown between groups in the SLJ (p = 0.001; d = 0.94) and the VJ (p = 0.001; d = 0.66). SLJ data in 1969 were higher (1.52 m, SD = 0.19) when compared to the 2016 data (1.34 m, SD = 0.18). The VJ performance of the 1969 sample was also higher (25.95 cm; SD = 6.58) than the 2016 sample (21.56 cm; SD = 4.72). SLJ and VJ performance of the 2016 group decreased 11.8% and 16.9%, respectively. There were no significant differences between groups in body mass index. The results indicate a secular decline in lower body muscular power in 10–11-year-old Spanish boys with no significant changes in body mass index over the 47-year study period.

A major obstacle for chimeric antigen receptor (CAR) T cell therapy in solid tumors is the lack of truly tumor-specific target antigens, which translates to the targeting of tumor-associated antigens (TAAs) overexpressed on tumors but shared with normal organs, raising safety concerns. In addition, expression of TAAs in solid tumors is particularly heterogeneous. In this regard, it is critical to deeply understand the sensitivity of CAR T cells, especially against low-density targets and the possible therapeutic window of antigen density targeted by CAR T cells. In this review, we discuss the recent findings of mechanisms of antigen recognition through CAR, including immunological synapse formation, and the impact of target antigen density for induction of distinct T cell functions. We also discuss rational strategies to adjust and expand the therapeutic window for effective and safe targeting of solid tumors by CAR T cell platforms.

Lexical Functional Grammar (LFG) uses abstract syntactic representation (f-structures) that tend to provide less hierarchical structure for certain constructions than those employed in other formal frameworks. This produces some good results, such as a very straightforward account of feature-sharing between phrases and their heads, but also certain difficulties, especially in cases where the semantic interpretation seems to be determined by the hierarchical c-structure rather than the flatter f-structure. These are unproblematic for all other major generative frameworks, but have been troublesome for standard versions of LFG. Here I will consider two such cases: scoping adjectival modification in noun phrases; and Romance `complex' (or `restructuring') predicates. Problems with the semantic interpretation of these constructions were first discussed by Andrews (1983) and Alsina (1997), respectively, and by others subsequently. Both constructions exhibit the problem of apparent concentricity, and a fully satisfactory and accepted LFG solution has not yet been found. My proposal is to use the hybrid objects and distribution convention of Dalrymple and Kaplan (2000), but with singleton rather than multi-member sets, along with a facility to stipulatively suppress distribution in individual constructions. This provides an analysis which explains scope-determination and helps with certain other problems, with far less change to the theory than in previous attempts such as Andrews and Manning (1993, 1999).

Integrative neuromuscular training (INT) has emerged as an effective strategy for improving health- and skill-related components of physical fitness, yet few studies have explored the cardiometabolic demands of this type of training in children. The aim of this study was to examine the acute cardiometabolic responses to a multi-modal INT protocol and to compare these responses to a bout of moderate-intensity treadmill (TM) walking in children. Participants (n = 14, age 10.7 ± 1.1 years) were tested for peak oxygen uptake (VO2) and peak heart rate (HR) on a maximal TM test and subsequently participated in two experimental conditions on nonconsecutive days: a 12-min INT protocol of six different exercises performed twice for 30 s with a 30 s rest interval between sets and exercises and a 12-min TM protocol of walking at 50% VO2peak. Throughout the INT protocol mean VO2 and HR increased significantly from 14.9 ± 3.6 mL∙kg−1∙min−1 (28.2% VO2 peak) to 34.0 ± 6.4 mL∙kg−1∙min−1 (64.3% VO2 peak) and from 121.1 ± 9.0 bpm (61.0% HR peak) to 183.5 ± 7.9 bpm (92.4% HR peak), respectively. While mean VO2 for the entire protocol did not differ between INT and TM, mean VO2 and HR during selected INT exercises and mean HR for the entire INT protocol were significantly higher than TM (all Ps ≤ 0.05). These findings suggest that INT can pose a moderate to vigorous cardiometabolic stimulus in children and selected INT exercises can be equal to or more metabolically challenging than TM walking.

Nasal colonization of methicillin-resistant Staphylococcus aureus (MRSA) plays an important role in the epidemiology and pathogenesis of disease. Situations of close-quarter contact in groups are generally regarded as a risk factor for community acquired MRSA strains due to transmission via fomites and person to person contact. With these criteria for risk, homeless individuals using shelter facilities, including showers and toilets, should be considered high risk for colonization and infection. The aim of this study was to determine the prevalence of nasal colonization of MRSA in a homeless population compared to established rates of colonization within the public and a control group of subjects from a neighboring medical school campus, and to analyze phylogenetic diversity among the MRSA strains. Nasal samples were taken from the study population of 332 adult participants, and analyzed. In addition, participants were surveyed about various lifestyle factors in order to elucidate potential patterns of behavior associated with MRSA colonization. Homeless and control groups both had higher prevalence of MRSA (9.8% and 10.6% respectively) when compared to the general population reported by previous studies (1.8%). However, the control group had a similar MRSA rate compared to healthcare workers (4.6%) while the homeless population had an increased prevalence. Risk factors identified in this study included male gender, age over 50 years and use of antibiotics within the past 3 months. Phylogenetic relationships between 9 of the positive samples from the homeless population were analyzed, showing 8 of the 9 samples had a high degree of relatedness between the spaA genes of the MRSA strains. This indicates that the same MRSA strain might be transmitted from person to person among homeless population. These findings increase our understanding of key differences in MRSA characteristics within homeless populations as well as risks for MRSA associated with being homeless, such as age and gender, which may then be a useful tool in guiding more effective prevention, treatment, and healthcare for homeless individuals.

Los efectos de la actividad física durante 12 semanas y el protocolo de cambio de conducta relacionada con la salud (Youth Fit For Life) llevados a cabo en los formatos de cuidados extraescolares durante 3 días/semana y educación física de 2 días/semana fueron evaluados y contrastados con niños afroamericanos de 9 a 12 años de edad, en un estudio experimental. El Índice de Masa Corporal, el porcentaje de grasa corporal y la fuerza muscular han mejorado significativamente en ambos formatos en chicos y chicas, con mayores efectos en la condición de cuidado extraescolar. Las mejorías significativas en estado de ánimo y auto-valoración fueron también asociadas a participación en el protocolo en ambos formatos con los tamaños de efectos algo mayores en el formato de educación física. El incremento de días por semana de actividad física voluntaria, moderada a vigorosa, fue significativo y similar en ambas condiciones (Mcambio = 0,71 y 0,75 días/semana, ps < 0,01). El análisis de regresión múltiple indicó que los cambios en el aspecto físico, autoconcepto físico, autoeficacia en ejercicios y la percepción del ser en general explicó una parte significativa de la varianza en los cambios en las sesiones de actividad física voluntaria a lo largo de 12 semanas, R2 = 0,24 a 0,73, ps < 0,01. Se sugiere implicaciones para el desarrollo de un modelo explicativo adecuado de la actividad física en niños, el diseño de la intervención en actividad física basado en la evidencia y reducción de obesidad en preadolescentes.

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline, in drug resistance, and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide, including CD53, CD10, EVI2B, and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally, we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological, therapeutic, and diagnostic understanding of myeloma.

CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.

Chimeric antigen receptor-T (CAR-T) cell therapies can eliminate relapsed and refractory tumors, but the durability of antitumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, and influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28- based CAR-T cells and changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing toward Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and antitumor activity. This finding suggests modifications to the costimulatory domains of CAR-T cells can enable longer persistence and thereby improve antitumor response., Introduction Chimeric antigen receptor-T (CAR-T) cell therapies mediate astonishing remissions of hematopoietic malignancies (1-3) and, in a few cases, solid tumors (4, 5). However, the antitumor response can be lost [...]

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).