IMPORTANCE Little is known about the penetration and bioactivity of systemically administered programmed cell death 1 (PD-1) antibodies in the central nervous system. Such information is critical for advancing checkpoint antibody therapies for treatment of brain tumors. OBJECTIVE To evaluate pembrolizumab concentrations and PD-1 blockade on T cells in the cerebrospinal fluid (CSF) after intravenous administration. DESIGN, SETTING, AND PARTICIPANTS Cerebrospinal fluid and blood samples were collected from 10 adult patients with high-grade gliomas who were participating in clinical trials of intracranially administered chimeric antigen receptor (CAR) T cells and intravenous pembrolizumab at City of Hope in Duarte, California, from 2017 through 2019. Neuropharmacokinetic and immunologic correlative studies were performed on CSF and serum samples. INTERVENTIONS OR EXPOSURES Pembrolizumab, 200 mg, was given intravenously every 3 weeks with a median of 2 cycles (range, 1-8). CAR T cells were administered intracranially every 1 to 4 weeks. Cerebrospinal fluid and blood samples were collected on the day of CAR T-cell administration and then 24 hours later for a total of 100 paired samples. MAIN OUTCOMES AND MEASURES Pembrolizumab concentrations were measured by enzyme-linked immunosorbent assay, PD-1 blocking on T cells by flow cytometry, and results of PD-1 blockade on CAR T-cell function by in vitro tumor rechallenge assays. RESULTS Of the 10 patients included in this study, the mean (SD) age was 45.7 (11.0) years, and 6 (60%) were women. Steady-state pembrolizumab concentrations in the CSF were achieved by 24 hours after initial intravenous administration, with a mean CSF:serum ratio of 0.009 (95% CI, 0.004-0.014). The CSF concentrations of pembrolizumab effectively blocked PD-1 on both endogenous T cells and intracranially administered CAR T cells in the CSF, with flow cytometric detection of surface PD-1 on the T cells decreasing from a mean (SD) of 39.3% (20.2%) before pembrolizumab to a mean (SD) of 3.8% (5.8%) 24 hours after pembrolizumab infusion. Steady-state concentrations in the CSF were maintained throughout the 21-day cycle of pembrolizumab, as was the PD-1 blocking effect, evidenced by no increase in detectable surface PD-1 on T cells in the CSF during that time period. Incubation of PD-1-expressing T cells with CSF samples from patients treated with pembrolizumab also resulted in PD-1 blockade. CONCLUSIONS AND RELEVANCE Results of this study demonstrate steady-state concentrations of pembrolizumab in CSF after intravenous administration as well as CSF concentrations that are sufficient for blocking PD-1 on endogenous and adoptively transferred T cells. This provides mechanistic insight regarding the ability of systemically administered PD-1 blocking antibodies to modulate T-cell activity in the brain.
Background Wide-spread application of chimeric antigen receptor (CAR) T cell therapy for cancer is limited by the current use of autologous CAR T cells necessitating the manufacture of individualized therapeutic products for each patient. To address this challenge, we have generated an off-the-shelf, allogeneic CAR T cell product for the treatment of glioblastoma (GBM), and present here the feasibility, safety and therapeutic potential of this approach. Methods We generated for clinical use a healthy-donor derived IL13Rα2-targeted CAR+ (IL13-zetakine+) cytolytic T-lymphocyte (CTL) product genetically engineered using zinc finger nucleases (ZFNs) to permanently disrupt the glucocorticoid receptor (GR) (GRm13Z40-2) and endow resistance to glucocorticoid treatment. In a phase I safety and feasibility trial we evaluated these allogeneic GRm13Z40-2 T cells in combination with intracranial administration of recombinant human IL-2 (rhIL-2; aldesleukin) in six patients with unresectable recurrent GBM that were maintained on systemic dexamethasone (4-12 mg/day). Results The GRm13Z40-2 product displayed dexamethasone-resistant effector activity without evidence for in vitro alloreactivity. Intracranial administration of GRm13Z40-2 in four doses of 10 8 cells over a two-week period with aldesleukin (9 infusions ranging from 2500 – 5000 IU) was well tolerated, with indications of transient tumor reduction and/or tumor necrosis at the site of T cell infusion in four of the six treated research subjects. Antibody reactivity against GRm13Z40-2 cells was detected in the serum of only one of the four tested subjects. Conclusions This first-in-human experience establishes a foundation for future adoptive therapy studies using off-the-shelf, zinc-finger modified and/or glucocorticoid resistant CAR T cells.
A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.
Although target cell cytolysis has been widely employed to describe effector function of cells, cytolysis assays as commonly employed do not generate quantitative data. In this report we describe the development and application of a statistically supported flow cytometry-based assay to quantify cell-mediated cytolysis. The assay depends on the use of the fluorescent dye CFSE to distinguish target from effector cells, the DNA intercalating dye 7AAD to distinguish dead from live cell events, and on the establishment of a cytolysis curve that allows for the derivation of statistically robust data. We demonstrate that the cytolysis curve is well described by a four parameter logistic regression model provided that (i) the range of effector to target (E:T) ratios studied allows for full description of the logistic curve, and (ii) an adequate number of data points are collected to estimate the model parameters. We show that the assay is highly reproducible and accurate, and comparable in sensitivity with the standard 51 Cr assay. We report on the potential for this assay to generate quantitative data on the cytolytic activity of both CD8 T and NK cells; describe a relationship between the efficiency of effector cell degranulation and target cell cytolysis throughout a range of E:T ratios, and demonstrate the potential to multiplex with other platforms to obtain broader datasets for the effector phenotype of cells. Appropriate use of this assay will enhance the ability to derive quantitative and integrated correlative datasets from basic, translational, and clinical studies. ' The most commonly employed assay to measure target cell lysis is the 51 Cr release assay (1). Although the 51 Cr release assay has enjoyed wide use and has provided useful data in a large number of clinical and basic research studies, the assay suffers from certain intrinsic limitations. Specifically, (i) the use of a radioactive element ( 51 Cr) with attendant issues of licensing, isotope half-life, storage, disposal, and compliance, (ii) the requirement for metabolic labeling, since metabolically slow cells can label very poorly, and cells at different metabolic states may be differentially resistant to lysis, and (iii) despite the fact that cytolysis is reported in the context of effector:target cell ratios (E:T), the read-out from 51 Cr-based assays is not based on a per-cell measurement but rather on bulk release of radioactivity into the culture medium.
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