Manufacturing of patient specific novel T cell therapies using the Cocoon® Platform automated system

Engineered T cell therapies, particularly chimeric antigen receptor T cell (CAR-T) immunotherapies, have proved effective against hematologic cancers. However, CAR-T therapies can potentiate immune responses causing cytokine release syndrome (CRS; “cytokine storm”) leading to adverse events in patients. Additionally, CAR-T has shown sporadic success in solid tumor indications. Novel therapies which activate T cells via the native T cell receptors (TCR) have shown greater tumor antigen recognition providing an alternative therapy which may prove effective against solid tumors. Utilizing novel cell immunotherapy modalities is only part of the solution as challenges remain to scale manufacturing to meet commercial demand. Scaling out commercial patient-specific cell therapy manufacturing for large populations using current methods will be expensive (cleanrooms and FTEs) and complex (logistics). Innovative manufacturing solutions will be required to manufacture patient-specific therapies in a robust and cost-effective manner. The Cocoon® Platform is one such innovation, a functionally-closed, automated, scalable cell therapy manufacturing platform. This abstract highlights a therapeutic T cell process translated from an open, manual process to the Cocoon® Platform. During process translation, the functionally-closed Cocoon® Platform was used to automate cell seeding, activation, transduction, feeding, real-time process monitoring, washing, and final product harvest using the single-use Cocoon cassette. During process development and translation, important process parameters were identified, optimized, and programmed enabling multiple process step automation removing the need for manual intervention. For the process, 200 million CD4+ and CD8+ isolated T cells were inoculated with TransActTM activator. The following day, cells were transduced with HER-2 lentivirus vector at various multiplicities of infection (MOI). Cells were expanded with a predefined feeding strategy in media supplemented with IL-2 until final product harvest. Following harvest, cells were assessed for cell yield, viability, transduction efficiency, and VCN. T cell phenotype and functionality was assessed via flow cytometry. The Cocoon manufacturing processes yielded 2.7 x 109 viable cells on average with viability \u3e85%. The Cocoon processes supported both CD4+ and CD8+ T cell expansion with 68% CD4+ T cells and 31% CD8+ T cells on average. The final product exhibited high T cell purity and viability (i.e. \u3e90% abTCR+ and 89% abTCR+, respectively) with transduction efficiencies varied from ~30% to \u3e65% depending on the process MOI. Vector copy number (VCN) was evaluated after each process and found to be ≤5 copies/transduced T cell. In summary, a gene-modified T cell process was successfully translated to the Cocoon and the harvested final products met all pre-defined acceptable criteria. The Cocoon represents a tool for manufacturing cell therapies in a robust manner, while maintaining comparability, and lowering manufacturing costs via increased automation. Ultimately the Cocoon will enable and accelerate development of cell therapies to address solid tumor indications and meet a critical patient need

Elimination de polluants des déchets liquides d’une unité de production de sucre par des argiles naturelles de Côte d’Ivoire

L’impact des déchets liquides industriels sur l’environnement demeure une réalité et une menace pour la qualité des eaux souterraines et de surfaces. Parmi les méthodes de dépollution existantes, l’adsorption des polluants par les argiles naturelles reste une méthode moins couteuse, disponible et facilement utilisable. La plupart des études sur l’aptitude des argiles à éliminer les polluants est faite avec des déchets liquides artificiels dont les concentrations sont maitrisées. L’objectif de cette étude était d’éliminer les polluants dans les déchets liquides d’une unité industrielle agronomique (UIA) de production de sucre par deux argiles naturelles de Côte d’Ivoire. Trois sources de déchets liquides provenant des activités de laboratoire d’analyse des sols, de jus de canne et de canne à sucre de l’UIA. Le traitement aux argiles a montré des taux d’élimination à divers degrés des polluants. Les paramètres suivants ont été mesuré avant et après le traitement aux argiles : pH, conductivité, turbidité, phosphore total, azote total, cuivre, zinc, plomb et mercure. L’argile de Katiola présente des aptitudes plus élevées à éliminer les polluants, comparée à l’argile d’Anyama. Cette étude confirme l’intérêt de l’utilisation des argiles pour la dépollution.Mots clés : Eaux usées, métaux lourds, pollution, argiles, adsorption.   English title: Removal of liquid waste pollutants from a sugar production unit using natural clays from Côte d'IvoireThe impact of industrial liquid waste on the environment remains a reality and a threat to groundwater and surface water quality. Among the existing depollution methods, the pollutants adsorption on natural clays remains a less expensive, available and easily usable method. Most studies on the ability of clays to remove pollutants are done with artificial liquid wastes whose concentrations are controlled. The objective of this study was to eliminate the pollutants in the liquid waste of an industrial agronomic sugar production unit using two natural clays from Côte d'Ivoire. Three sources of liquid wastes from the UIA's analysis laboratory activities. Treatment with clays showed varying degrees of removal of pollutants. The following parameters were measured before and after the clay treatment: pH, conductivity, turbidity, total suspended solids, total phosphorus, total nitrogen, copper, zinc, lead and mercury. The values of these parameters are compared to the national standard. Katiola clay exhibits higher abilities to remove pollutants compared to Anyama clay. This study confirms the value of using clays for depollution.Keywords: Wastewater, heavy metals, pollution, clays, adsorption

Evidence for Active Control of Rectus Extraocular Muscle Pulleys

PURPOSE. Connective tissue structures constrain paths of the rectus extraocular muscles (EOMs), acting as pulleys and serving as functional EOM origins. This study was conducted to investigate the relationship of orbital and global EOM layers to pulleys and kinematic implications of this anatomy. METHODS. High-resolution magnetic resonance imaging (MRI) was used to define the anterior paths of rectus EOMs, as influenced by gaze direction in living subjects. Pulley tissues were examined at cadaveric dissections and surgical exposures. Human and monkey orbits were step and serially sectioned for histologic staining to distinguish EOM fiber layers in relationship to pulleys. RESULTS. MRI consistently demonstrated gaze-related shifts in the anteroposterior locations of human EOM path inflections, as well as shifts in components of the pulleys themselves. Histologic studies of human and monkey orbits confirmed gross examinations and surgical exposures to indicate that the orbital layer of each rectus EOM inserts on its corresponding pulley, rather than on the globe. Only the global layer of the EOM inserts on the sclera. This dual insertion was visualized in vivo by MRI in human horizontal rectus EOMs. CONCLUSIONS. The authors propose the active-pulley hypothesis: By dual insertions the global layer of each rectus EOM rotates the globe while the orbital layer inserts on its pulley to position it linearly and thus influence the EOM's rotational axis. Pulley locations may also be altered in convergence. This overall arrangement is parsimoniously suited to account for numerous aspects of ocular dynamics and kinematics, including Listing's law. (Invest Ophthalmol Vis Sci. 2000;41: 1280 -1290 I nitial attempts to mathematically model binocular alignment showed the importance to extraocular muscle (EOM) action of EOM paths and the pivotal mechanical role of orbital connective tissues. The need for EOM path data motivated early radiographic studies in monkeys 1 and humans, 2 suggesting that paths of rectus EOMs are stabilized relative to the orbit. A decade ago, Miller 3 used relatively low-resolution MRI with three-dimensional (3-D) reconstruction to demonstrate stability of rectus EOM belly paths throughout the oculomotor range

Evidence for Active Control of Rectus Extraocular Muscle Pulleys

PURPOSE. Connective tissue structures constrain paths of the rectus extraocular muscles (EOMs), acting as pulleys and serving as functional EOM origins. This study was conducted to investigate the relationship of orbital and global EOM layers to pulleys and kinematic implications of this anatomy. METHODS. High-resolution magnetic resonance imaging (MRI) was used to define the anterior paths of rectus EOMs, as influenced by gaze direction in living subjects. Pulley tissues were examined at cadaveric dissections and surgical exposures. Human and monkey orbits were step and serially sectioned for histologic staining to distinguish EOM fiber layers in relationship to pulleys. RESULTS. MRI consistently demonstrated gaze-related shifts in the anteroposterior locations of human EOM path inflections, as well as shifts in components of the pulleys themselves. Histologic studies of human and monkey orbits confirmed gross examinations and surgical exposures to indicate that the orbital layer of each rectus EOM inserts on its corresponding pulley, rather than on the globe. Only the global layer of the EOM inserts on the sclera. This dual insertion was visualized in vivo by MRI in human horizontal rectus EOMs. CONCLUSIONS. The authors propose the active-pulley hypothesis: By dual insertions the global layer of each rectus EOM rotates the globe while the orbital layer inserts on its pulley to position it linearly and thus influence the EOM's rotational axis. Pulley locations may also be altered in convergence. This overall arrangement is parsimoniously suited to account for numerous aspects of ocular dynamics and kinematics, including Listing's law. (Invest Ophthalmol Vis Sci. 2000;41: 1280 -1290 I nitial attempts to mathematically model binocular alignment showed the importance to extraocular muscle (EOM) action of EOM paths and the pivotal mechanical role of orbital connective tissues. The need for EOM path data motivated early radiographic studies in monkeys 1 and humans, 2 suggesting that paths of rectus EOMs are stabilized relative to the orbit. A decade ago, Miller 3 used relatively low-resolution MRI with three-dimensional (3-D) reconstruction to demonstrate stability of rectus EOM belly paths throughout the oculomotor range

Effector and Central Memory Poly-Functional CD4+ and CD8+ T Cells are Boosted upon ZOSTAVAX® Vaccination

ZOSTAVAX® is a live attenuated varicella-zoster virus (VZV) vaccine that is licensed for the protection of individuals ≥ 50 years against shingles, and its most common complication, post-herpetic neuralgia. While IFN responses increase upon vaccination, the quality of the T cell response has not been elucidated. By using polychromatic flow cytometry, we characterized the breadth, magnitude, and quality of ex vivo CD4+ and CD8+ T cell responses induced 3 – 4 weeks after ZOSTAVAX vaccination of healthy adults. We show, for the first time that the highest frequencies of VZV-specific CD4+ T cells were poly-functional CD154+IFNγ+IL-2+TNFα+ cells, which were boosted upon vaccination. The CD4+ T cells were broadly reactive to several VZV proteins, with IE63 ranking the highest amongst them in the fold-rise of poly-functional cells, followed by IE62, gB, ORF9, and gE. We identified a novel poly-functional ORF9-specific CD8+ T cell population in 62% of the subjects, and these were boosted upon vaccination. Poly-functional CD4+ and CD8+ T cells produced significantly higher levels of IFNγ, IL-2, and TNFα compared to mono-functional cells. After vaccination, a boost in the expression of IFN by poly-functional IE63-and ORF9-specific CD4+ T cells, and IFNγ, IL-2, and TNFα by ORF9-specific poly-functional CD8+ T cells was observed. Responding poly-functional T cells exhibited both effector (CCR7−CD45RA−CD45RO+), and central (CCR7+CD45RA−CD45RO+) memory phenotypes, which expressed comparable levels of cytokines. Altogether, our studies demonstrate that a boost in memory poly-functional CD4+ T cells, and ORF9-specific CD8+ T cells may contribute towards ZOSTAVAX efficacy

Quasi-three-level Model Applied to Measured Spectra of Nonlinear Absorption and Refraction in Organic Molecules

Materials with a large nonlinear refractive index (2) and relatively small linear and nonlinear absorption losses, namely, two-photon absorption (2PA, of coefficient 2), have long been sought after for applications such as all-optical switching (AOS). Here we experimentally determine the linear and 2PA properties of several organic molecules, which we approximate as centrosymmetric, and use a simplified essential-state model (quasi-three-level model) to predict the dispersion of 2. We then compare these predictions with experimental measurements of 2 and find good agreement. Here “quasi”-three-level means using a single one-photon allowed intermediate state and multiple (here two) two-photon allowed states. This also allows predictions of the figure-of-merit (FOM), defined as the ratio of nonlinear refractive phase shift to the 2PA fractional loss, that determines the viability for such molecules to be used in device applications. The model predicts that the optimized wavelength range for a large FOM lies near the short wavelength linear absorption edge for cyanine-like dyes where the magnitude of 2 is quite large. However, 2PA bands lying close to the linear absorption edge in certain classes of molecules can greatly reduce this FOM. We identify two molecules having a large FOM for AOS. We note that the FOM is often defined as the ratio of real to imaginary parts of the third-order susceptibility ((3)) with multiple processes leading to both components. As explained later in this paper, such definitions require care to only include the 2PA contribution to the imaginary part of (3) in regions of transparency.Abstract © 2016 Optical Society of Americ

Detection of Gamma-rays around 1TeV from RX J0852.0-4622 by CANGAROO-II

We have detected gamma-ray emission at the 6sigma level at energies greater than 500GeV from the supernova remnant RX J0852.0-4622 (G266.2-1.2) using the CANGAROO-II Imaging Atmospheric Cherenkov Telescope (IACT). The flux was 0.12 times of that of Crab at 1TeV. The signal centroid is consistent with the peak of the X-ray emission in the north-west rim of the remnant.Comment: 12pages, 4figures, to be published in ApJ

An 800-million-solar-mass black hole in a significantly neutral Universe at redshift 7.5

Quasars are the most luminous non-transient objects known and as a result they enable studies of the Universe at the earliest cosmic epochs. Despite extensive efforts, however, the quasar ULAS J1120+0641 at z=7.09 has remained the only one known at z>7 for more than half a decade. Here we report observations of the quasar ULAS J134208.10+092838.61 (hereafter J1342+0928) at redshift z=7.54. This quasar has a bolometric luminosity of 4e13 times the luminosity of the Sun and a black hole mass of 8e8 solar masses. The existence of this supermassive black hole when the Universe was only 690 million years old---just five percent of its current age---reinforces models of early black-hole growth that allow black holes with initial masses of more than about 1e4 solar masses or episodic hyper-Eddington accretion. We see strong evidence of absorption of the spectrum of the quasar redwards of the Lyman alpha emission line (the Gunn-Peterson damping wing), as would be expected if a significant amount (more than 10 per cent) of the hydrogen in the intergalactic medium surrounding J1342+0928 is neutral. We derive a significant fraction of neutral hydrogen, although the exact fraction depends on the modelling. However, even in our most conservative analysis we find a fraction of more than 0.33 (0.11) at 68 per cent (95 per cent) probability, indicating that we are probing well within the reionization epoch of the Universe.Comment: Updated to match the final journal versio

ZNF804a Regulates Expression of the Schizophrenia-Associated Genes PRSS16, COMT, PDE4B, and DRD2

ZNF804a was identified by a genome-wide association study (GWAS) in which a single nucleotide polymorphism (SNP rs1344706) in ZNF804a reached genome-wide statistical significance for association with a combined diagnosis of schizophrenia (SZ) and bipolar disorder. Although the molecular function of ZNF804a is unknown, the amino acid sequence is predicted to contain a C2H2-type zinc-finger domain and suggests ZNF804a plays a role in DNA binding and transcription. Here, we confirm that ZNF804a directly contributes to transcriptional control by regulating the expression of several SZ associated genes and directly interacts with chromatin proximal to the promoter regions of PRSS16 and COMT, the two genes we find upregulated by ZNF804a. Using immunochemistry we establish that ZNF804a is localized to the nucleus of rat neural progenitor cells in culture and in vivo. We demonstrate that expression of ZNF804a results in a significant increase in transcript levels of PRSS16 and COMT, relative to GFP transfected controls, and a statistically significant decrease in transcript levels of PDE4B and DRD2. Furthermore, we show using chromatin immunoprecipitation assays (ChIP) that both epitope-tagged and endogenous ZNF804a directly interacts with the promoter regions of PRSS16 and COMT, suggesting a direct upregulation of transcription by ZNF804a on the expression of these genes. These results are the first to confirm that ZNF804a regulates transcription levels of four SZ associated genes, and binds to chromatin proximal to promoters of two SZ genes. These results suggest a model where ZNF804a may modulate a transcriptional network of SZ associated genes