CAR-T cell expansion platforms yield distinct T cell differentiation states.

With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4+CD8+ cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7+CD45RA+ naïve/stem central memory (Tn/scm)-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O2 from day 5 onward, we could generate >60% Tn/scm-like cells, with longer time in hypoxia correlating with a higher percentage of Tn/scm-like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells.

Establishment and validation of in-house cryopreserved CAR/TCR-T cell flow cytometry quality control.

BACKGROUND: Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available. Therefore, we established a procedure to generate an in-house cryopreserved CAR/TCR T-cell products for use as a flow cytometry quality control and validated their use. METHODS: Two CAR T-cell products: CD19/CD22 bispecific CAR T-cells and FGFR4 CAR T-cells and one TCR-engineered T-cell product: KK-LC-1 TCR T-cells were manufactured in Center for Cellular Engineering (CCE), NIH Clinical Center. The products were divided in aliquots, cryopreserved and stored in the liquid nitrogen. The cryopreserved flow cytometry quality controls were tested in flow cytometry assays which measured post-thaw viability, CD3, CD4 and CD8 frequencies as well as the transduction efficiency and vector identity. The long-term stability and shelf-life of cryopreserved quality control cells were evaluated. In addition, the sensitivity as well as the precision assay were also assessed on the cryopreserved quality control cells. RESULTS: After thawing, the viability of the cryopreserved CAR/TCR T-cell controls was found to be greater than 50%. The expression of transduction efficiency and vector identity markers by the cryopreserved control cells were stable for at least 1 year; with post-thaw values falling within ± 20% range of the values measured at time of cryopreservation. After thawing and storage at room temperature, the stability of these cryopreserved cells lasted at least 6 h. In addition, our cryopreserved CAR/TCR-T cell quality controls showed a strong correlation between transduction efficiency expression and dilution factors. Furthermore, the results of flow cytometric analysis of the cryopreserved cells among different laboratory technicians and different flow cytometry instruments were comparable, highlighting the reproducibility and reliability of these quality control cells. CONCLUSION: We developed and validated a feasible and reliable procedure to establish a bank of cryopreserved CAR/TCR T-cells for use as flow cytometry quality controls, which can serve as a quality control standard for in-process and lot-release testing of CAR/TCR T-cell products.

Antibody screening using a human iPSC-based blood-brain barrier model identifies antibodies that accumulate in the CNS.

Drug delivery across the blood-brain barrier (BBB) remains a significant obstacle for the development of neurological disease therapies. The low penetration of blood-borne therapeutics into the brain can oftentimes be attributed to the restrictive nature of the brain microvascular endothelial cells (BMECs) that comprise the BBB. One strategy beginning to be successfully leveraged is the use of endogenous receptor-mediated transcytosis (RMT) systems as a means to shuttle a targeted therapeutic into the brain. Limitations of known RMT targets and their cognate targeting reagents include brain specificity, brain uptake levels, and off-target effects, driving the search for new and potentially improved brain targeting reagent-RMT pairs. To this end, we deployed human-induced pluripotent stem cell (iPSC)-derived BMEC-like cells as a model BBB substrate on which to mine for new RMT-targeting antibody pairs. A nonimmune, human single-chain variable fragment (scFv) phage display library was screened for binding, internalization, and transcytosis across iPSC-derived BMECs. Lead candidates exhibited binding and internalization into BMECs as well as binding to both human and mouse BBB in brain tissue sections. Antibodies targeted the murine BBB after intravenous administration with one particular clone, 46.1-scFv, exhibiting a 26-fold increase in brain accumulation (8.1 nM). Moreover, clone 46.1-scFv was found to associate with postvascular, parenchymal cells, indicating its successful receptor-mediated transport across the BBB. Such a new BBB targeting ligand could enhance the transport of therapeutic molecules into the brain.

Sickle Cell Anemia Mediates Carotid Artery Expansive Remodeling That Can Be Prevented by Inhibition of JNK (c-Jun N-Terminal Kinase).

OBJECTIVE: Sickle cell anemia (SCA) causes chronic inflammation and multiorgan damage. Less understood are the arterial complications, most evident by increased strokes among children. Proteolytic mechanisms, biomechanical consequences, and pharmaceutical inhibitory strategies were studied in a mouse model to provide a platform for mechanistic and intervention studies of large artery damage due to sickle cell disease. Approach and Results: Townes humanized transgenic mouse model of SCA was used to test the hypothesis that elastic lamina and structural damage in carotid arteries increased with age and was accelerated in mice homozygous for SCA (sickle cell anemia homozygous genotype [SS]) due to inflammatory signaling pathways activating proteolytic enzymes. Elastic lamina fragmentation observed by 1 month in SS mice compared with heterozygous littermate controls (sickle cell trait heterozygous genotype [AS]). Positive immunostaining for cathepsin K, a powerful collagenase and elastase, confirmed accelerated proteolytic activity in SS carotids. Larger cross-sectional areas were quantified by magnetic resonance angiography and increased arterial compliance in SS carotids were also measured. Inhibiting JNK (c-jun N-terminal kinase) signaling with SP600125 significantly reduced cathepsin K expression, elastin fragmentation, and carotid artery perimeters in SS mice. By 5 months of age, continued medial thinning and collagen degradation was mitigated by treatment of SS mice with JNK inhibitor. CONCLUSIONS: Arterial remodeling due to SCA is mediated by JNK signaling, cathepsin proteolytic upregulation, and degradation of elastin and collagen. Demonstration in Townes mice establishes their utility for mechanistic studies of arterial vasculopathy, related complications, and therapeutic interventions for large artery damage due to SCA.

Cutaneous reactions in children treated with MEK inhibitors, BRAF inhibitors, or combination therapy: A multicenter study.

BACKGROUND: Treatment with BRAF inhibitors (BRAFI) and MEK inhibitors (MEKI) causes cutaneous reactions in children, limiting dosing or resulting in treatment cessation. The spectrum and severity of these reactions is not defined. OBJECTIVE: To determine the frequency and spectrum of cutaneous reactions in children receiving BRAFI and MEKI and their effects on continued therapy. METHODS: A multicenter, retrospective study was conducted at 11 clinical sites in the United States and Canada enrolling 99 children treated with BRAFI and/or MEKI for any indication from January 1, 2012, to January 1, 2018. RESULTS: All children in this study had a cutaneous reaction; most had multiple, with a mean per patient of 3.5 reactions on BRAFI, 3.7 on MEKI, and 3.4 on combination BRAFI/MEKI. Three patients discontinued treatment because of a cutaneous reaction. Treatment was altered in 27% of patients on BRAFI, 39.5% on MEKI, and 33% on combination therapy. The cutaneous reactions most likely to alter treatment were dermatitis, panniculitis, and keratosis pilaris-like reactions for BRAFI and dermatitis, acneiform eruptions, and paronychia for MEKI. CONCLUSIONS: Cutaneous reactions are common in children receiving BRAFI and MEKI, and many result in alterations or interruptions in oncologic therapy. Implementing preventative strategies at the start of therapy may minimize cutaneous reactions.

Continuing patient care to underserved communities and medical education during the COVID-19 pandemic through a teledermatology student-run clinic.

A virtual pediatric dermatology student-run clinic was initiated during the COVID-19 pandemic, when in-person educational opportunities were limited. The clinic's aim is to provide high-quality dermatologic care to a diverse, underserved pediatric patient population while teaching trainees how to diagnose and manage common skin conditions. In our initial eight sessions, we served 37 patients, predominantly those with skin of color, and had a low no-show rate of 9.8%. This report describes the general structure of the clinic, goals, and the patient population to provide an overview of our educational model for those interested in similar efforts.

Age-dependent characterization of carotid and cerebral artery geometries in a transgenic mouse model of sickle cell anemia using ultrasound and microcomputed tomography.

To define morphological changes in carotid and cerebral arteries in sickle cell transgenic mice (SS) as they age, a combination of ultrasound and microcomputed tomography of plastinated arteries was used to quantify arterial dimensions and changes in mice 4, 12, and 24 weeks of age. 12-week SS mice had significantly larger common carotid artery diameters than AS mice, which continued through to the extracranial and intracranial portions of the internal carotid artery (ICA). There were also side specific differences in diameters between the left and right vessels. Significant ICA tapering along its length occurred by 12- and 24-weeks in SS mice, decreasing by as much as 70%. Significant narrowing along the length was also measured in SS anterior cerebral arteries at 12- and 24-weeks, but not AS. Collectively, these findings indicate that sickle cell anemia induces arterial remodeling in 12- and 24-weeks old mice. Catalog of measurements are also provided for the common carotid, internal carotid, anterior cerebral, and middle cerebral arteries for AS and SS genotypes, as a reference for other investigators using mathematical and computational models of age-dependent arterial complications caused by sickle cell anemia.

Velocity influences the relative contributions of visual and vestibular cues to self-acceleration.

Self-motion perception is based on the integration of visual (optic flow) and vestibular (inertial) sensory information. Previous research has shown that the relative contribution of visual and vestibular cues can change in real time based on the reliability of that information. The present study assessed whether initial velocity and acceleration magnitude influence the relative contribution of these cues to the detection of self-acceleration. Participants performed a simple response time task with visual and vestibular self-acceleration cues as targets. Visual optic flow was presented at three possible initial velocities of 3, 9, or 15 m/s, and accelerated to result in three possible final velocities of 21, 27, or 33 m/s. Corresponding vestibular cues were presented at magnitudes between 0.01 and 0.04 g. The self-acceleration cues were presented at three possible stimulus onset asynchronies (SOAs): visual-first (by 100 ms), in-sync, and vestibular-first (by 100 ms). We found that presenting the cues in-sync resulted in the fastest responses across all velocities and acceleration magnitudes. Interestingly, presenting the visual cue first resulted in a relative advantage over vestibular-first at the slowest initial velocity of 3 m/s, and vice versa for the fastest initial velocity of 15 m/s. The fastest overall responses for visual-first and in-sync were observed at 9 m/s. The present results support the hypothesis that velocity of optic flow can alter the relative contribution of visual and vestibular cues to the detection of self-acceleration.

Cutaneous reactions to targeted therapies in children with CNS tumors: A cross-sectional study.

BACKGROUND: MAPK (RAS-RAF-MEK-ERK-MAP) and mTOR inhibitors are novel treatments for pediatric central nervous system (CNS) tumors. The literature on common cutaneous adverse reactions to these therapies is sparse in the pediatric population. The aim of this study was to describe common cutaneous adverse reactions to BRAF, MEK, and mTOR inhibitors in children with CNS tumors. METHODS: In this cross-sectional study, patients younger than 21 years of age receiving BRAF, MEK, and mTOR inhibitor monotherapy for a CNS tumor were enrolled over a one-year period. Full body skin examination, photographs of dermatologic findings, and initial treatment recommendations were included at the initial visit, and follow-up skin examinations were recommended every three months. RESULTS: Twenty-two patients were enrolled in the study. Fifty percent (11/22) received trametinib, a MEK inhibitor, 27.3% (6/22) received dabrafenib, a BRAF inhibitor, and 22.7% (5/22) received everolimus, an mTOR inhibitor. Median age at visit was 11 years (range, 3-19). Median time from treatment initiation to skin examination was 4.5 months (range, 0-43). Ninety-six percent (21/22) of all patients had at least one skin reaction. The most common reactions across treatment groups included follicular/acneiform eruptions and xerosis. Two patients on MEK inhibitors and one patient on a BRAF inhibitor required therapy cessation due to severe cutaneous reactions. CONCLUSIONS: Cutaneous reactions to targeted anticancer therapy in children are common, treatable, and rarely require drug dose reduction or discontinuation. Routine surveillance and early intervention may improve quality of life and facilitate continuation of life-saving therapy.

Impact of skin biopsy on the management of acute graft-versus-host disease in a pediatric population.

BACKGROUND/OBJECTIVES: Acute graft-versus-host disease (GVHD) of the skin is a common complication of hematopoietic stem cell transplantation (HSCT) but often represents a diagnostic challenge. The adult literature suggests that histopathology rarely dictates management decisions, but the clinical utility of skin biopsies in pediatric patients with suspected acute GVHD is unknown. The objective of this study was to determine the frequency with which skin biopsy leads to a definitive diagnosis of acute GVHD and changes the management of acute GVHD in the pediatric population. METHODS: We conducted a retrospective analysis of histopathology results and the associated impact on clinical management based on chart review of pediatric patients who underwent skin biopsy for cutaneous eruptions suspicious for acute GVHD from 1995 to 2016. RESULTS: Among 27 pediatric HSCT patients, skin biopsy yielded definitive diagnoses (GVHD or otherwise) in only 15% (4/27) of cases. Overall, dermatology consultation was associated with clinical management changes in 78% (21/27) of cases. A change in management was definitively based on skin biopsy results in only 7.4% (2/27) of cases. The mean duration of time between dermatology consultation and return of biopsy results was 4.8 days (range 1-17). CONCLUSIONS: Our results suggest that skin biopsy of pediatric HSCT patients with findings concerning for acute skin GVHD rarely yields a definitive diagnosis or change in management.

Improvement of hereditary palmoplantar keratoderma with oral trametinib.

We report a child with a past medical history notable for congenital deafness, palmoplantar keratoderma (PPK), and hypothalamic glioma who initiated a MEK inhibitor trametinib for cancer-directed therapy at 11 years of age and was incidentally noted to have marked improvement in his PPK. Trametinib withdrawal led to worsening in the patient's PPK. We speculate that the patient's PPK improved because of trametinib, given the temporal relationship between trametinib therapy and PPK severity, observed both after introduction and withdrawal of trametinib therapy. The upregulation of MAPK signaling may be involved in the pathogenesis of keratinocyte proliferation in at least some forms of PPK, given that downstream inhibition of MAPK signaling led to an improvement in the patient's PPK.

Wound infection after inpatient pediatric skin biopsy.

Punch and shave biopsies are important dermatologic procedures in the inpatient setting but have a risk of wound infection. Data on the incidence of infection in this setting are limited and conflicting. This retrospective study of 127 pediatric inpatients at two tertiary care centers who underwent biopsy demonstrated a low overall risk of infection (n = 1, 0.8%). Twenty-five (19.7%) were neutropenic, and 51 (40.2%) were not taking systemic antibiotics at the time of biopsy; none of these patients developed a wound infection. The overall low rate of infection should reassure physicians who are balancing the risks and benefits of performing a skin biopsy in children in the inpatient setting and suggests that physicians should not defer clinically indicated biopsies because of concern about infection.

Herpes zoster at the vaccination site in immunized healthy children.

In this case series, we report seven immunized healthy children without underlying immunodeficiency who presented with herpes zoster that correlated with varicella-zoster vaccination site. The morphology of the lesions included erythematous papules, pseudovesicles, and plaques, with associated pain in two and pruritus in three patients; systemic symptoms ranged from none to low-grade fevers, upper respiratory symptoms, and joint pain. These cases highlight the clinical, diagnostic, and therapeutic implications of herpes zoster in vaccinated children.

Outpatient dermatology consultation impacts the diagnosis and management of pediatric oncology patients: A retrospective study.

BACKGROUND: The impact of dermatology consultation on the care of children with oncologic conditions is unknown. OBJECTIVE: To review outpatient dermatology visits and the resulting impact on diagnosis and management of pediatric oncology patients. METHOD: Retrospective review of pediatric oncology patients with outpatient dermatology visits at a tertiary care center from 2008 to 2015. RESULTS: The most common dermatologic diagnoses in 516 patients were skin infections (21.3%) and nonmalignant skin eruptions (33.4%). A diagnosis of significant impact (ie, malignancy, adverse cutaneous drug reaction, graft-versus-host disease, varicella-zoster virus, or herpes simplex virus infection), was made at the dermatology clinic in 14.7% of visits. Consultation resulted in a change in diagnosis in 59.8% of patients, change in dermatologic management in 72.4% of patients, and change in management of noncutaneous issues in 12.4% of patients. LIMITATIONS: The use of electronic medical records, the nongeneralizable study population, and the retrospective design represent potential limitations. CONCLUSION: Outpatient dermatology consultation can affect the care of pediatric oncology patients with respect to diagnosis and treatment of skin conditions and management of nondermatologic issues.

Primary Cutaneous B-Cell Lymphoblastic Lymphoma Arising from a Long-Standing Lesion in a Child and Review of the Literature.

Precursor B-cell lymphoblastic lymphoma (B-LBL) is a rare entity and primary cutaneous B-LBL is an even more uncommon diagnosis that typically affects children. A 4-year-old boy presented with a persistent rash on his left cheek for almost 2 years and was found to have primary cutaneous B-LBL. We report this case to emphasize that B-LBL should be in the differential diagnosis for an otherwise unimpressive persistent lesion in the head and neck region and review all reported pediatric cases of primary cutaneous B-LBL without extracutaneous involvement.

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures.

We describe derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension culture-reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor-expressing cells based on their differential survival and proliferation in suspension culture. Seamless integration of SiPSC reprogramming and directed differentiation enabled scalable production of beating cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step toward the development of robust PSC generation, expansion and differentiation technology.

Surfaceome profiling reveals regulators of neural stem cell function.

The composition of cell-surface proteins changes during lineage specification, altering cellular responses to their milieu. The changes that characterize maturation of early neural stem cells (NSCs) remain poorly understood. Here we use mass spectrometry-based cell surface capture technology to profile the cell surface of early NSCs and demonstrate functional requirements for several enriched molecules. Primitive NSCs arise from embryonic stem cells upon removal of Transforming growth factor-ß signaling, while definitive NSCs arise from primitive NSCs upon Lif removal and FGF addition. In vivo aggregation assays revealed that N-cadherin upregulation is sufficient for the initial exclusion of definitive NSCs from pluripotent ectoderm, while c-kit signaling limits progeny of primitive NSCs. Furthermore, we implicate EphA4 in primitive NSC survival signaling and Erbb2 as being required for NSC proliferation. This work elucidates several key mediators of NSC function whose relevance is confirmed on forebrain-derived populations and identifies a host of other candidates that may regulate NSCs.