Characterization of the input material quality for the production of tisagenlecleucel by multiparameter flow cytometry and its relation to the clinical outcome.

Tisagenlecleucel (tisa-cel) is a CD19-specific CAR-T cell product approved for the treatment of relapsed/refractory (r/r) DLBCL or B-ALL. We have followed a group of patients diagnosed with childhood B-ALL (n = 5), adult B-ALL (n = 2), and DLBCL (n = 25) who were treated with tisa-cel under non-clinical trial conditions. The goal was to determine how the intensive pretreatment of patients affects the produced CAR-T cells, their in vivo expansion, and the outcome of the therapy. Multiparametric flow cytometry was used to analyze the material used for manufacturing CAR-T cells (apheresis), the CAR-T cell product itself, and blood samples obtained at three timepoints after administration. We present the analysis of memory phenotype of CD4/CD8 CAR-T lymphocytes (CD45RA, CD62L, CD27, CD28) and the expression of inhibitory receptors (PD-1, TIGIT). In addition, we show its relation to the patients' clinical characteristics, such as tumor burden and sensitivity to prior therapies. Patients who responded to therapy had a higher percentage of CD8+CD45RA+CD27+ T cells in the apheresis, although not in the produced CAR-Ts. Patients with primary refractory aggressive B-cell lymphomas had the poorest outcomes which was characterized by undetectable CAR-T cell expansion in vivo. No clear correlation of the outcome with the immunophenotypes of CAR-Ts was observed. Our results suggest that an important parameter predicting therapy efficacy is CAR-Ts' level of expansion in vivo but not the immunophenotype. After CAR-T cells' administration, measurements at several timepoints accurately detect their proliferation intensity in vivo. The outcome of CAR-T cell therapy largely depends on biological characteristics of the tumors rather than on the immunophenotype of produced CAR-Ts.

Biodeterioration of Compost-Pretreated Polyvinyl Chloride Films by Microorganisms Isolated From Weathered Plastics.

Polyvinyl chloride (PVC) is a petroleum-based plastic used in various applications, polluting the environment because of its recalcitrance, large content of additives, and the presence of halogen. In our case study, a new, two-stage biodegradation technology that combined composting process used for PVC pretreatment with a subsequent PVC attack by newly-isolated fungal and bacterial strains under SSF conditions was used for biodegradation of commercial PVC films. The novelty consisted in a combined effect of the two biodegradation processes and the use for augmentation of microbial strains isolated from plastic-polluted environments. First, the ability of the newly-isolated strains to deteriorate PVC was tested in individual, liquid-medium- and SSF cultures. Higher mass-reductions of PVC films were obtained in the former cultures, probably due to a better mass transfer in liquid phase. Using the two-stage biodegradation technology the highest cumulative mass-reductions of 29.3 and 33.2% of PVC films were obtained after 110 days with Trichoderma hamatum and Bacillus amyloliquefaciens applied in the second stage in the SSF culture, respectively. However, FTIR analysis showed that the mass-reductions obtained represented removal of significant amounts of additives but the PVC polymer chain was not degraded.

Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells.

The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3' and 5' arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 108 CAR19 T cells per one electroporation of 1 × 107 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products.

Kinetics of Resorcinol-Formaldehyde Condensation-Comparison of Common Experimental Techniques.

Porous carbons, originated from resorcinol-formaldehyde (RF) gels, show high application potential. However, the kinetics and mechanism of RF condensation are still not well described. In this work, different methods (dynamic light scattering-DLS, Fourier transform infrared spectroscopy-FTIR, low field 1H nuclear magnetic resonance relaxometry-1H-NMR, and differential scanning calorimetry-DSC) were used to follow the isothermal RF condensation of mixtures varying in catalyst content (Na2CO3) and reactant concentration. The applicability and results obtained by the methods used differ significantly. The changes in functional groups can be followed by FTIR only at very early stages of the reaction. DLS enables the estimate of the growth of particles in reaction solution, but only before the solution becomes more viscous. Following the relaxation of 1H nuclei in water during RF condensation brings a different view on the system-this technique follows the properties of the present water that is gradually captured in polymeric gel. From this side, the process behaves similarly to the nucleation reaction, which is in contradiction to the n-order mechanism confirmed by other techniques. The widest range of applicability was found for DSC measurement of the freezing/melting behavior of the reaction mixture, which is possible to use without any limitations until full solidification. Furthermore, this approach enables us to follow the gradual formation and development of the gel through the intermediate undergoing glass transition.

Inducible secretion of IL-21 augments anti-tumor activity of piggyBac-manufactured chimeric antigen receptor T cells.

BACKGROUND: The efficiency of chimeric antigen receptor (CAR) T-cell-based therapies depends on a sufficient expansion of CAR T cells in vivo and can be weakened by intra-tumoral suppression of CAR T cell functions, leading to a failure of therapy. For example, certain B-cell malignancies such as chronic lymphocytic leukemia are weakly sensitive to treatment with CAR T cells. Co-expression of proinflamatory cytokines such as IL-12 and IL-18 by CAR T cells have been shown to enhance their antitumor function. We similarly engineered CAR T cell to co-express IL-21 and studied the effects of IL-21 on CAR T cells specific to CD19 and prostate-specific membrane antigens using an in vitro co-culture model and NSG mice transplanted with B-cell tumors. RESULTS: IL-21 enhanced the expansion of CAR T cells after antigenic stimulation, reduced the level of apoptosis of CAR T cells during co-culture with tumor cells and prevented differentiation of CAR T cells toward late memory phenotypes. In addition, induced secretion of IL-21 by CAR T cells promoted tumor infiltration by CD19-specific CAR (CAR19) T cells in NSG mice, resulting in reduced tumor growth. By co-culturing CAR19 T cells with bone-marrow fragments infiltrated with CLL cells we demonstrate that IL-21 reduces the immunosupressive activity of CLL cells against CAR19 T cells. CONCLUSIONS: CAR19 T cells armed with IL-21 exhibited enhanced antitumor functions. IL-21 promoted their proliferation and cytotoxicity against chronic lymphocytic leukemia (CLL). The results suggest that arming CAR T cells with IL-21 could boost the effectiveness of CAR T-mediated therapies.

Interaction among clays and bovine serum albumin.

Interactions between bovine serum albumin and various clays including pure clay minerals and bentonite were studied with the aim to describe the interaction process. The adsorption of albumin on the clays is strongly affected by the behavior of clays in the aquatic environment (hydrolysis and release of cations). A sufficient amount of albumin was adsorbed on the acid-activated montmorillonite K10 (0.067 mg mg-1) and on the illite-smectite (0.086 mg mg-1). These clay minerals do not strongly affect the sorption solution parameters such as pH value and content of cations. Practically no adsorption was observed on the bentonite and vermiculite. Bentonite and vermiculite are subject to stronger interactions with water which cause the increase of pH value of the sorption solution and release of cations to the solution and thus they cause conformational changes of albumin, which was confirmed by circular dichroism measurements. Obtained results were confirmed by infrared spectroscopy and thermal analysis as well. Interaction of studied materials with bovine serum albumin causes the reduction of particle size in the case of all studied clays except vermiculite. Albumin probably attacks the clay structure during the adsorption, which causes the decrease of particle size. The presented work contributes to the knowledge about interaction of bovine serum albumin with clays in the field of influence of physico-chemical behaviour of clays in the solution on the interaction with albumin.

Conference report: Isranalytica 2012.

The Isranalytica, organized by the Israel Analytical Chemical Society, is the largest analytical conference in Israel and has become one of the largest annual conferences in analytical chemistry in the world. Over the past years, Isranalytica has achieved major success and become the most prestigious analytical chemistry meeting in Israel.

Interior and interfacial aqueous solvation of benzene dicarboxylate dianions and their methylated analogues: A combined molecular dynamics and photoelectron spectroscopy study.

Aqueous solvation of benzene dicarboxylate dianions (BCD(2-)) was studied by means of photoelectron spectroscopy and molecular dynamics simulations. Photoelectron spectra of hydrated o- and p-BCD(2-) with up to 25 water molecules were obtained. An even-odd effect was observed for the p-BCD(2-) system as a result of the alternate solvation of the two negative charges. However, the high polarizability of the benzene ring makes the two carboxylate groups interact with each other in p-BCD(2-), suppressing the strength of this even-odd effect compared with the linear dicarboxylate dianions linked by an aliphatic chain. No even-odd effect was observed for the o-BCD(2-) system, because each solvent molecule can interact with the two carboxylate groups at the same time due to their proximity. For large solvated clusters, the spectral features of the solute decreased while the solvent features became dominant, suggesting that both o- and p-BCD(2-) are situated in the center of the solvated clusters. Molecular dynamics simulations with both nonpolarizable and polarizable force fields confirmed that all three isomers (o-, m-, and p-BCD(2-)) solvate in the aqueous bulk. However, upon methylation the hydrophobic forces overwhelm electrostatic interactions and, as a result, the calculations predict that the tetramethyl-o-BCD(2-) is located at the water surface with the carboxylate groups anchored in the liquid and the methylated benzene ring tilted away from the aqueous phase.

Unified molecular picture of the surfaces of aqueous acid, base, and salt solutions.

The molecular structure of the interfacial regions of aqueous electrolytes is poorly understood, despite its crucial importance in many biological, technological, and atmospheric processes. A long-term controversy pertains between the standard picture of an ion-free surface layer and the strongly ion specific behavior indicating in many cases significant propensities of simple inorganic ions for the interface. Here, we present a unified and consistent view of the structure of the air/solution interface of aqueous electrolytes containing monovalent inorganic ions. Molecular dynamics calculations show that in salt solutions and bases the positively charged ions, such as alkali cations, are repelled from the interface, whereas the anions, such as halides or hydroxide, exhibit a varying surface propensity, correlated primarily with the ion polarizability and size. The behavior of acids is different due to a significant propensity of hydronium cations for the air/solution interface. Therefore, both cations and anions exhibit enhanced concentrations at the surface and, consequently, these acids (unlike bases and salts) reduce the surface tension of water. The results of the simulations are supported by surface selective nonlinear vibrational spectroscopy, which reveals among other things that the hydronium cations are present at the air/solution interface. The ion specific propensities for the air/solution interface have important implications for a whole range of heterogeneous physical and chemical processes, including atmospheric chemistry of aerosols, corrosion processes, and bubble coalescence.

Structure and vibrational spectroscopy of salt water/air interfaces: predictions from classical molecular dynamics simulations.

We report the sum frequency generation (SFG) spectra of aqueous sodium iodide interfaces computed with the methodology outlined by Morita and Hynes (J. Phys. Chem. B 2002, 106, 673), which is based on molecular dynamics simulations. The calculated spectra are in qualitative agreement with experiment. Our simulations show that the addition of sodium iodide to water leads to an increase in SFG intensity in the region of 3400 cm(-1), which is correlated with an increase in ordering of hydrogen-bonded water molecules. Depth-resolved orientational distribution functions suggest that the ion double layer orders water molecules that are approximately one water layer below the Gibbs dividing surface. We attribute the increase in SFG intensity to these ordered subsurface water molecules that are present in the aqueous sodium iodide/air interfaces but are absent in the neat water/air interface.

Enhanced concentration of polarizable anions at the liquid water surface: SHG spectroscopy and MD simulations of sodium thiocyanate [corrected].

Contrasting current textbook descriptions, a consistent picture of substantial concentration enhancement of highly polarizable anions at the surface of aqueous electrolyte solutions is emerging. Such enhancement may have important implications for chemistry occurring on aqueous aerosols and ocean surfaces. Here we present a combined experimental and theoretical investigation of the liquid/air interface of aqueous sodium thiocyanate [corrected] at varying salt concentrations. Normalized second harmonic generation intensities fitted to Langmuir isotherms yield a Gibbs free energy of adsorption of -1.80 kcal/mol. These results are in accord with molecular dynamics simulations in slab geometry, which predict an appreciable surface enhancement of SCN-.

Bulk versus interfacial aqueous solvation of dicarboxylate dianions.

Solvation of dicarboxylate dianions of varying length of the aliphatic chain in water clusters and in extended aqueous slabs was investigated using photoelectron spectroscopy and molecular dynamics simulations. Photoelectron spectra of hydrated succinate, adipate, and tetradecandioic dianions with up to 20 water molecules were obtained. Even-odd effects were observed as a result of the alternate solvation mode of the two negative charges with increasing solvent numbers. The competition between hydrophilic interactions of the charged carboxylate groups and hydrophobic interactions of the aliphatic chain leads to conformation changes in large water clusters containing dicarboxylates bigger than adipate. It also leads to a transition from bulk aqueous solvation of small dicarboxylates to solvation at the water/vapor interface of the larger ones. Whereas oxalate and adipate solvate in the inner parts of the aqueous slab, suberate and longer dicarboxylate dianions have a strong propensity to the surface. This transition also has consequences for the folding of the flexible aliphatic chain and for the structure of aqueous solvation shells around the dianions.

Solvent-mediated folding of a doubly charged anion.

The microsolvation of the suberate dianion, -O2C(CH2)6CO2-, with two separate charge centers was studied by photoelectron spectroscopy and molecular dynamics simulation one solvent molecule at a time for up to 20 waters. It is shown that the two negative charges are solvated in the linear suberate alternately. As the solvent number increases, the negative charges are screened and a conformation change occurs at 16 waters, where the cooperative hydrogen bonding of water is large enough to overcome the Coulomb repulsion and pull the two negative charges closer through a water bridge. This conformation change, revealed both from the experiment and from the simulation, is a manifestation of the hydrophilic and hydrophobic forces at the molecular level.